Protein-based biomaterials exhibiting viscoelastic behavior, processes for obtaining same and uses thereof - Patent Application 20070122997

Abstract

The present invention relates to a process for preparing a biomaterial, the process comprising the steps of: a) preparing a solution containing at least one protein having a solubility in water equal to or greater than about 10 mg / mL and at least one salt having a solubility in water equal to or greater than about 500 mg / mL; and b) evaporating the solution obtained in step a) either neat, as a foam obtained by foaming the solution obtained in step a), or as a mixture thereof, at a temperature comprised between 4 and 50°C at atmospheric pressure, or at a lower temperature under vacuum, or at a pressure lower than atmospheric pressure, until two immiscible phases are formed or a substantially dry solid is obtained, thereby obtaining a biomaterial. The present invention also relates to the biomaterial obtained by the process and to its use as a support for in vitro tissue engineering, and / or for in vitro cell culture and in vitro growth, and / or as an implantable medical device, or as a drug.

Description

[Technical Field]

[0001] The present invention refers to a process for the preparation of a biomaterial, the biomaterial obtained by said process, and the use of the biomaterial as a support for tissue engineering, for cell culture or growth, as an implantable medical device, and as a drug.

[0002] Thus, the present invention has utility in the medical field, particularly in tissue engineering, drug delivery, wound dressings and implants.

[0003] In the following description, references in brackets ([]) refer to the list of references at the end of the text. [Background technology]

[0004] Biomaterials are widely used in a variety of therapeutic applications, including tissue engineering, drug delivery, wound dressings, and transplants.

[0005] Synthetic, biological or hybrid, they have been increasingly used for decades in all therapeutic areas: cardiovascular, surgical and orthopedic, dental, ophthalmological, dermatological, urological, nephrological, neurological, endocrinological, especially to regenerate or improve tissue function.

[0006] Although there are several types of biomaterials, four main categories of biomaterials can be envisaged. metals and metal alloys, Ceramics in the broad sense, Polymers and soft matter, Coral or other components extracted from plant or animal organisms, such as naturally occurring materials, for example chitin, alginate, heparin, fucoidan, cellulose, collagen or fibrin.

[0007] This last category of biomaterials is of particular interest because naturally derived materials are generally naturally biocompatible and biodegradable.

[0008] Based on naturally occurring materials, several techniques, including thermal aggregation or crosslinking, have been used to prepare biomaterials. However, the use of high temperatures and crosslinking agents can cause irreversible denaturation and changes in the structure of the material, resulting in the loss of its biological properties and potentially revealing new antigenic sites that can trigger inflammatory responses.

[0009] Therefore, there is a need for alternative biomaterials based on naturally derived materials that do not have these drawbacks and that meet the need for more natural biomaterials. Summary of the Invention

[0010] Through extensive research, applicants have developed new biomaterials that exhibit outstanding mechanical properties and excellent stability in water, acidic solutions, and cell culture media.

[0011] Surprisingly, the resulting biomaterial behaves like a solid and is suitable as a support for tissue engineering or cell culture.

[0012] This disclosure reports the first biomaterial made from perhaps a completely unique type of protein, or a few selected proteins that are not denatured during the biomaterial preparation process. This means that the biomaterial of the present invention is unlikely to induce an inflammatory response in subjects. Furthermore, this biomaterial is non-cytotoxic and favors cell adhesion and colonization.

[0013] Because it is made of protein, the biomaterial of the present invention is completely biodegradable.

[0014] The preparation process developed by the applicant allows obtaining biomaterials without the use of chemical or harsh / denaturing compounding conditions. It also allows for the adjustment of biomaterial properties, particularly mechanical and intrinsic properties, to obtain functionalized biomaterials with new properties. Thus, the applicant provides versatile biomaterials whose properties can be adjusted to meet the requirements of the targeted therapeutic application.

[0015] Thus, in a first aspect, the present invention provides a process for the preparation of a biomaterial comprising the steps of: a) preparing a solution comprising at least one protein having a water solubility equal to or greater than about 10 mg / mL and at least one salt having a water solubility equal to or greater than about 500 mg / mL. b) evaporating the solution obtained in step a) either neat, as a foam obtained by foaming the solution obtained in step a), or as a mixture thereof, at a temperature comprised between 4 and 50°C at atmospheric pressure, or at a lower temperature under vacuum, or at a pressure lower than atmospheric pressure, until two immiscible phases are formed or a substantially dry solid is obtained, thereby obtaining a biomaterial.

[0016] The process of the present invention has the important advantage of not using a covalent cross-linking or thermal aggregation step, thereby resulting in a biomaterial with undenatured proteins.

[0017] The at least one protein used in step a) can be any protein with a solubility in water of about 10 mg / mL or greater at 20°C, e.g., 20 mg / mL, 40 mg / mL, or 50 mg / mL or greater. For example, the protein can have a solubility of about 10 mg / mL to 1000 mg / mL. The solubility of the protein in water can be measured by any method known in the art, such as high-performance liquid chromatography (HPLC), attenuated total reflectance (ATR)-FTIR spectroscopy, Raman spectroscopy, or focused beam reflectance mode (FBRM) measurement. Such proteins can be selected from serum proteins such as albumin or globulins, particularly gamma-globulin. Albumins can be selected from human serum albumin, bovine serum albumin, porcine serum albumin, ovalbumin, plant albumin, and recombinant albumin, such as recombinant human albumin produced in rice, which is structurally equivalent to native human serum albumin. Albumin can also be albumin nanoparticles. As noted above, "at least one" protein can be used in the process of the present invention, meaning that one, two, three, four, or more different proteins can be used in the solution of step a). Preferably, one to three different proteins can be used. The protein concentration in the solution of step a) can be any concentration that allows mixing with salt, including, for example, a range of 10 mg / mL to 500 mg / mL. Those skilled in the art can adapt the protein concentration in the solution depending on the nature and concentration of the salt. However, as explained in more detail below, the molar ratio of salt to protein is more important than the protein concentration to obtain a biomaterial with the desired function.

[0018] The at least one salt of step a) has a concentration equal to or greater than about 500 mg / mL at a temperature of 20° C., e.g. For example, the salt may be any salt with a water solubility of equal to or greater than 700 mg / mL, 900 mg / mL, or 1200 mg / mL. For example, the salt may have a solubility between about 500 mg / mL and 3000 mg / mL. The water solubility of the salt can be measured at a temperature of about 20°C, pH 7, and atmospheric pressure. The water solubility of the salt can be measured by any method known to those skilled in the art, such as ion chromatography, gas chromatography, acid-base titration, potentiometric titration, volumetric measurement, weighing, or Raman spectroscopy. The at least one salt may be selected from, for example, NaBr, NaI, KI, CaCl2, MgCl2, KC2H3O2, and NH4HCO2. As indicated above, "at least one" salt can be used in the process of the present invention, meaning that one, two, three, four, or more different salts can be used. Preferably, one to three salts can be used. For example, the at least one salt may comprise NaBr and NaI, NaBr and CaCl2, KI and CaCl2, or NaBr / CaCl2 / MgCl2, e.g., in molar ratios of 100 / 100 / 100, 200 / 200 / 200, or 300 / 300 / 300. The salt concentration in the solution in step a) may be any concentration that allows mixing with the protein. It may range from 0.01 M to 40 M, for example. Those skilled in the art can adjust the salt concentration in the solution depending on the nature and concentration of the protein. However, as explained in more detail below, the molar ratio of salt to protein is more important than the salt concentration to obtain a biomaterial with the desired function.

[0019] Preferably, the molar ratio of the at least one protein and the at least one salt in step a) depends on the nature of the protein and the nature of the salt used to obtain the biomaterial. Because the applicant has demonstrated that biomaterial formation depends on the combined effect of both protein and salt concentrations, the salt / protein molar ratio is a more appropriate and reliable parameter for evaluating membrane formation. Knowing this, the molar ratio can be determined without undue burden by a person skilled in the art, taking into account their general knowledge and the desired properties of the insoluble biomaterial, particularly its rigidity. For example, the molar ratio may range from 100 to 4000, e.g., 100 to 3000, or 300 to 2500, or 400 to 2000, or 600 to 1500, or 650 to 1000, depending on the salt and protein used in step a). For example, the molar ratio of a mixture of NaBr and albumin may be 664.

[0020] The solution of step a) can be achieved by mixing at least one protein and at least one salt in a suitable solvent or mixture of solvents under non-denaturing conditions. The solvent can be selected without undue burden by a person skilled in the art, taking into account general knowledge and the properties of the salt and protein. For example, the solvent can be selected from water, a buffer solution such as acetate buffer, a mixture of water and buffer, and another water-miscible solvent such as ethanol, methanol, acetone, DMF, or DMSO. The temperature of the solution during step a) can be between 5°C and 40°C.

[0021] Step a) can be carried out at any pH that avoids protein denaturation known to those skilled in the art. Preferably, step a) is carried out at a pH between 3.0 and 9.0, including, optionally excluding, the value of 3.0. The pH can be, for example, between 4.0 and 9.0, or between 4.0 and 8.0.

[0022] The mixture can be realized or transferred onto any container or support adapted to receive such a mixture. It can be, for example, a glass or silicone mold, a microscope or microarray substrate, a cell and tissue culture dish or a microwell plate, or a polymeric support around which the biomaterial can be shaped. Advantageously, the support can be selected taking into account the desired surface area, shape and thickness of the biomaterial to be obtained. For this purpose, the amount of mixture poured into the container can be determined by the surface area and / or thickness of the support. The thickness of the biomaterial may be selected depending on the desired thickness of the biomaterial. The biomaterial may have any shape, for example, a membrane, a solid or hollow cylinder, a cone, a sphere, or a paving stone. For reference, as exemplified below, the M / S ratio, which is the ratio between the initial weight of the protein used in the formulation and the area of ​​the container, is 10 mg / cm. 2 ~400mg / cm 2 , e.g., 20 mg / cm 2 ~400mg / cm 2 may include:

[0023] The evaporation step b) can be carried out on the solution obtained in step a) itself, either directly after step a) or after an intermediate step that does not change the properties or physical structure of the solution obtained in step a).

[0024] Alternatively, step b) can be carried out on the foam obtained by foaming the solution obtained in step a). The foam can be obtained, for example, by applying mechanical work to the solution obtained in step a) to increase the surface area of ​​the solution. This can be done by any method known to those skilled in the art, for example, by stirring, by dispersing a large amount of gas into the solution obtained in step a), or by injecting gas into the solution obtained in step a).

[0025] In another embodiment, step b) can be performed on a mixture of the solution obtained in step a) and a foam obtained by foaming the solution obtained in step a). In this case, a portion of the solution obtained in step a) can be taken and foamed in a separate container. The foam obtained can be evaporated directly or returned to the solution, mixed with it, and then evaporated as in step b). Preferably, gentle mixing is performed to preserve the foam. Evaporation of the foam produces a highly porous biomaterial.

[0026] The evaporation in step b) is carried out to allow the formation of two immiscible phases or to obtain a substantially dry solid. Advantageously, it is carried out under conditions that allow avoiding denaturation of the protein present in the solution, foam, or mixture thereof. To this end, the temperature and pressure can be determined and mutually adjusted to achieve this goal. Those skilled in the art can determine these parameters depending on the type of protein or salt and in accordance with their general knowledge. For example, the temperature can be 4 to 50°C at atmospheric pressure, such as 4°C to 20°C, or 10°C to 50°C, or 15°C to 50°C, or 20°C to 50°C, or 25 to 40°C, or 25 to 35°C, or 20 to 30°C. It is also possible to carry out step b) at a lower temperature under vacuum or at a pressure lower than atmospheric pressure. In this case, the temperature can be, for example, 1°C to 20°C, or 2°C to 15°C, or 5°C to 10°C, and the pressure can be 1 to 100 kPa. In either case, evaporation is carried out until two immiscible phases are formed or until a substantially dry solid is obtained. This is the case, for example, when a thin layer of salt is deposited on the surface of the biomaterial or when a solid containing less than 20% by weight of water, e.g., less than 10% water, is obtained. The formation of two immiscible phases or a substantially dry solid can be visually recognized. For reference, this can optionally be verified by measuring the water content by gravimetric analysis.

[0027] The duration of the evaporation step can be determined by those skilled in the art without undue burden, according to their general knowledge. It can be a function of the type of protein or salt, the temperature and pressure selected for carrying out the process, the amount of solution to be evaporated, or the shape of the container. For example, evaporation can be carried out for 10 hours to 30 days, e.g., 1 day to 20 days, or 2 days to 30 days. Longer periods are also possible, but often do not result in improved technical characteristics of the biomaterial.

[0028] Step b) can be carried out at any pH that avoids denaturation of the protein known to those skilled in the art. For example, step b) can be carried out at a pH between 3.0 and 9.0, including, optionally, excluding the value of 3.0. Depending on the type of protein, the pH can be, for example, It can be 4.0 to 9.0, or 4.0 to 8.0.

[0029] Evaporation can be carried out by any means that meets the above criteria, such as an oven or vacuum oven.

[0030] The process of the invention may consist of steps a) and b) as described above, making it possible to obtain the biomaterial of the invention, in which case there are no other steps in the process and the biomaterial can be obtained directly at the end of step b), as it may be a substantially dry solid or a solid phase of two immiscible phases obtained in step b).

[0031] Alternatively, the process of the invention may comprise additional steps that make it possible to obtain the biomaterial of the invention. The additional steps can be carried out before step a) and / or between steps a) and b) and / or after step b). In this case, the biomaterial can be obtained after carrying out these additional steps.

[0032] For example, the solid phase or dry solid obtained in step b) can be washed to remove at least a portion of the salts, thereby obtaining a biomaterial. Preferably, the solid phase or dry solid obtained in step b) can be washed until at least 90% by weight of the at least one salt is removed, thereby obtaining a biomaterial. Washing can be carried out, for example, until at least 95% or at least 99% by weight of the at least one salt is removed. Washing can be carried out by any means known to those skilled in the art, for example, using distilled water or an aqueous buffer solution. Control of the resulting salt concentration can be carried out by any known method, for example, by BCA or microanalysis, as shown below.

[0033] The soaking step of the solid phase or dry solid obtained in step b) can be carried out, for example, after washing. Soaking allows the biomaterial to be hydrated. Soaking can be carried out by any means known to those skilled in the art, for example, using distilled water or a buffer solution at room temperature for 48 hours.

[0034] It is also possible to add at least one additive during step a) and / or step b), or during any of the additional steps described above. The additive may be any substance that allows the properties of the biomaterial to be adjusted as desired. In some cases, the additive may also allow salts or portions thereof to be removed from the biomaterial. The additives can be selected by those skilled in the art according to general knowledge and the desired properties of the biomaterial. They may be selected, for example, from polymers, particularly uncharged, positively charged, negatively charged, and zwitterionic polymers, nonionic amino acids, and particles. The polymer may be any natural polymer selected from polysaccharides, proteins, peptides, and polynucleotides, and / or synthetic and semi-synthetic polymers. For example, polymers include, but are not limited to, polypeptides, homopolypeptides, chitosan, hyaluronic acid, heparin, alginate, chondroitin sulfate, polyarginine, polylysine, ε-polylysine, DEAE dextran, polycyclodextrin, polyallylamine hydrochloride, polyethyleneimine, xanthan gum, polyacrylic acid, polypeptide glycol, starch, cellulose and its derivatives, collagen, insulin, fibrinogen, casein, gelatin, gliadin, gluten, elastin, globulin, and hemoglobin. Amino acids can be selected from all suitable amino acids, preferably arginine, ornithine, lysine, and cysteine. Particles can be any suitable particles, such as nanoparticles, e.g., carbon nanotubes or graphene, microparticles, bacteria, and viral vectors. Additives can be incorporated by any means known to those skilled in the art. For example, it can be by directly dissolving and / or suspending the additive in the solution obtained in step a) or by dissolving and / or suspending the additive in a water-miscible solvent. The additive can be incorporated into the solution obtained in step a) by subsequently adding the mixture to the solution obtained in step a). Additionally or alternatively, the additive can be incorporated into the biomaterial obtained in step b) by adsorbing the additive dissolved and / or suspended in a solvent onto the biomaterial obtained in step b). The amount of additive can be adapted to the desired properties of the protein and biomaterial and can therefore be determined by those skilled in the art according to their general knowledge. For example, the percentage of additive can be 0 to 20% by weight, for example, 1 to 18% by weight, or 2 to 15% by weight, or 3 to 12% by weight, based on the total amount of protein in the biomaterial.

[0035] Additionally or alternatively, at least one active ingredient can be incorporated into the solution obtained in step a) and / or the biomaterial obtained in step b). This may enable functionalization of the biomaterial. The active ingredient can be incorporated by any means known to those skilled in the art. For example, it can be incorporated into the solution obtained in step a) by directly dissolving and / or suspending the active ingredient in the solution obtained in step a), or by dissolving and / or suspending the active ingredient in a water-miscible solvent and then adding the mixture to the solution obtained in step a). Additionally or alternatively, the active ingredient can be incorporated into the biomaterial obtained in step b) by adsorption of the active ingredient dissolved and / or suspended in the solvent to the biomaterial obtained in step b). In this case, the solvent can be selected from water, an organic solvent, or a mixture of water and a water-miscible solvent. Advantageously, up to 30% (v / v) of organic solvent can be added to the protein / salt solution without inhibiting membrane formation or significantly altering the properties of the prepared material, allowing the incorporation of water-insoluble active ingredients. For example, depending on the nature of the protein and solvent, the total organic solvent can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% (v / v). For example, it can be up to 15% ethanol, 10% DMSO, or 30% acetonitrile or dichloromethane. Those skilled in the art can adapt this amount to the protein, salt, and solvent characteristics by routine experimentation. Advantageously, it is possible to add up to 30% (v / v) of organic solvent to the BSA / salt solution without inhibiting film formation or significantly altering the properties of the prepared material, thus allowing the incorporation of water-insoluble active substances.

[0036] The active ingredient can be any substance that makes it possible to confer interesting properties on the biomaterial. The active ingredient can be selected by the person skilled in the art according to his general knowledge and the desired properties of the biomaterial. It may be selected from, but is not limited to, goserelin, leuprolide, carmustine, paclitaxel, histrelin, or gemcitabine, anti-inflammatory agents such as diclofenac, immunosuppressants such as azathioprine or methotrexate, immunomodulators such as cyclosporine, regulators of extracellular cells, cell proliferation inhibitors such as imatinib or axitinib, anticoagulants such as rivaroxaban or edoxaban, antithrombotic agents such as clopidogrel, enzyme inhibitors, analgesics such as morphine or hydrocodone, antiproliferative agents, antifungal substances, cytostatic substances, matrix-interacting thrombopoietin including growth factors such as erythropoietin, enzymes, hormones, steroids such as hydrocortisone or prednisolone, non-steroidal substances, and antihistamines such as diphenhydramine or fexofenadine, anticancer substances. The amount of active ingredient can be adapted to the desired properties of the protein and biomaterial and can therefore be determined by those skilled in the art according to their general knowledge. For example, the percentage of the active ingredient can be 0-30% by weight, for example, 0-25% by weight, or 1-25% by weight, or 1-20% by weight, or 1-18% by weight, or 2-15% by weight, or 3- It may be 12% by weight.

[0037] In any case, the biomaterial obtained by the process of the present invention may comprise at least 50% by weight, for example at least 55%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or even 100% protein, based on the total weight of the biomaterial.

[0038] The biomaterial obtained by carrying out the preparation process is a second object of the present invention.

[0039] As explained above, the properties of the biomaterial, including its visual appearance, can be adjusted by changing one or more of the parameters of the preparation process, in particular the type of salt and the salt / protein ratio, thereby offering the possibility to tailor the properties of the biomaterial as needed.

[0040] With regard to the hardness of the biomaterial, it can be a solid insoluble biomaterial ranging from foams to compact materials including hydrogels.

[0041] Regarding the shape of the biomaterial, it can be any desired shape depending on the intended use and the container used to prepare the biomaterial. It can be a membrane, a tube, a cylinder, a pad, a ring, but this list is not limiting. The biomaterial can also be cut after the process to obtain the desired shape or size.

[0042] The biomaterial can be made into any desired size, including particulates, by grinding the biomaterial.

[0043] The visual aspect of the biomaterial can be translucent to opaque.

[0044] As mentioned above, one of the main advantages of the biomaterial of the present invention is that the protein used to prepare the biomaterial is not denatured during the preparation process of the present invention. The preparation is carried out under non-denaturing conditions, and the shape and / or secondary structure of the protein can be analyzed in the solution obtained in step a) and the biomaterial obtained in step b). "Not denatured" means, according to the present invention, that the percentage of secondary structure of the protein in the solution obtained in step a) is at least equivalent to that of a control solution or control dry native protein powder of the corresponding native protein prepared at a similar concentration, and that the percentage of secondary structure of the protein in the biomaterial obtained in step b) shows at least a substantial increase in β-turns and intermolecular β-sheets, and a significant decrease in disordered structure, compared to the corresponding native protein. The shape and percentage of secondary structure of the protein in the biomaterial can be controlled by any method known to those skilled in the art, such as IR analysis or SAXS (small-angle X-ray scattering), as shown below.

[0045] The biomaterial of the present invention is particularly stable over time. Advantageously, it is stable in aqueous solutions at acidic, neutral, and basic pHs, and / or in organic solvents such as ethanol for at least 2 days, preferably at least 7 days. This means that there is essentially no dissolution of the biomaterial during this period, even though structural changes due to the disruption of hydrogen bonds and disulfide bridges and a mass loss of less than 20% are observed. For example, mass loss can be up to 20% in basic solutions and up to 10% in water, ethanol, and acidic solutions. Mass loss of the biomaterial can be calculated as shown in the examples below.

[0046] As explained above, biomaterials may be made exclusively of proteins, in particular non-denatured proteins, which means excellent biocompatibility. To improve the therapeutic properties, the composition may be associated with at least one active ingredient, as exemplified above.

[0047] Another object of the present invention therefore relates to the use of the biomaterials of the present invention as supports for in vitro tissue engineering and / or in vitro cell culture and growth and / or implantable medical devices. Indeed, the biomaterials of the present invention provide the structural and biochemical support necessary for cell growth, and because they may be three-dimensional, they are particularly suitable for cell culture and drug / cell delivery.

[0048] Another object of the present invention relates to the use of biomaterials for use as drugs. Because the biomaterials of the present invention can interact with biological systems, they can be used, for example, to construct diagnostic devices, tissue or organ substitutes, or functional replacement devices. Therefore, the biomaterials of the present invention can be used in vivo as implantable medical devices, particularly to replace defective tissue in subjects in need of such replacement, or as drug delivery systems. In other words, the present invention also describes implantable medical devices comprising the biomaterials of the present invention. Advantageously, devices or drugs comprising the biomaterials of the present invention can be absorbed in vivo after a certain period of time, depending on the properties of the biomaterial. For example, the period may be 20, 30, or more than 60 days after insertion into the body.

[0049] The present invention is further illustrated by the following examples, which should not be construed as limiting, with reference to the accompanying drawings. [Brief explanation of the drawings]

[0050] [Figure 1] This figure illustrates the preparation of an albumin-based biomaterial by evaporation in the presence of salt. The molar ratio of NaBr / BSA (bovine serum albumin) in the selected formulation is 664. Evaporation is continued in an oven (37 °C) at atmospheric pressure for 7 days until the biomaterial is completely dry. Excess salt forms a thin layer on the surface of the biomaterial. After evaporation, a washing and soaking process is applied to remove the salt, leaving behind a water-insoluble albumin-based biomaterial. [Figure 2]This shows the time course of BSA / NaBr664 film formation (evaporation at 37°C, pH=6, M / S=105 mg / cm2). Once washed, the film becomes transparent. The bottom of the mold is covered with a non-stick silicone disk. The irregularities in the upper right corner are due to air bubbles. [Figure 3] 1 shows the surface charge titration of albumin with increasing concentrations of NaBr (pH=6). [Figure 4] Rheological properties of albumin-based membranes are shown by an oscillation protocol (frequency ramp: 100 Hz to 0.01 Hz, shear stress control: 1 Pa). The shear modulus of the elastic component (Pa) is represented by the solid line, and the shear modulus of the viscous component (Pa) is represented by the dashed line. [Figure 5] Stability of BSA / NaBr664 films (evaporation at 37°C and pH=6) in various dissolution media (from left to right: water, saline, 1M NaCl solution, 1M NaBr solution, acidic solution pH 3, basic solution pH 10, ethanol, and trypsin 0.125 mg / mL). For each dissolution medium, a film was placed in 25 mL of medium. The experiment was carried out at 37°C with stirring for 7 days. [Figure 6] Distilled water [ka] , 2-mercaptoethanol [ka] and urea solution [ka] This figure shows the rheological properties of BSA / NaBr664 films incubated with ethanol (evaporation at 37 °C and pH = 6). For the solution, two batches of films were placed in 30 mL of medium. The experiment was carried out at room temperature for 24 h. The rheological properties of the films were evaluated by an oscillation protocol (frequency ramp: 100 Hz to 0.01 Hz, shear stress control: 1 Pa). [Figure 7A]Fitting of the mid-I band of albumin in BSA solution (100 mg / mL in DO) and identification of each secondary structure subband (the fitting curve and the original mid-I band spectrum overlap, residual RMS error <0.005). [Figure 7B] Comparison of the BSA mid-I band in a BSA control solution (100 mg / mL, DO, solid line) and a BSA / NaBr664 solution (BSA: 100 mg / mL, NaBr: 1 M, DO, dashed line). [Figure 7C] A comparison of the BSA mid-I band in a BSA control powder (solid line) and a BSA / NaBr664 film (dashed line) is shown. [Figure 7D] A comparison of the BSA mid-I band in a BSA control solution (100 mg / mL, D2O, solid line) and a BSA control powder (dashed line) is shown. [Figure 7E] Comparison of the middle I band of BSA in a BSA / NaBr664 solution (BSA: 100 mg / mL, NaBr: 1 M, DO, solid line) and a BSA / NaBr664 membrane (dashed line). [Figure 8A] Scattering curve of BSA solution (solution 2, 40 wt% HO) and theoretical intensity measured with CRYSOL using monomeric BSA protein from 3v03PBD atomic coordinates (50 harmonics, excluded volume 8.7104, default values ​​for other parameters). A scale factor was applied to fit the data to the theoretical curve. [Figure 8B] X-ray scattering curves of (a) dry powder of BSA, (b, c) solutions of BSA ((b) Solution 1 containing 24.21 wt% HO and (c) Solution 2 containing 39.09 wt% HO), (d) dry powder of NaBr (* indicates Bragg reflections at 111, 200, and 220), and (e) a BSA / NaBr664 film. Data are shifted vertically for clarity. [Figure 9A] Figure 1 shows the SEM analysis of an albumin-based membrane formulated at a molar ratio of NaBr / BSA of 664 under selected conditions (evaporation at 37°C and pH = 6). The sample was metallized before observation. The membrane surface. [Figure 9B]Figure 1 shows an SEM analysis of an albumin-based membrane formulated at a molar ratio of NaBr / BSA of 664 under selected conditions (evaporation at 37°C and pH = 6). The sample was metallized before observation. Cross section of the membrane. [Figure 10] Figure 1 represents the cell viability of Balbc3T3 fibroblasts treated with membrane extracts (12.5%, 25%, 50%, and 100% BSA / NaBr400, BSA / NaBr664, and BSA / CaCl2700). Indirect cytotoxicity was estimated by comparing the normalized metabolic activity of Balbc3T3 cells cultured for 24 hours in contact with BSA / NaBr400, BSA / NaBr664, and BSA / CaCl2700 extracts with that of the positive control (Ctl+). (*) A significant difference was observed between the treatment group and the positive control (Ctl+) (p<0.05). Biological replicate = 1, total technical replicates = 4. [Figure 11] Figure 1 shows the cell viability of Balbc 3T3 fibroblasts cultured in direct contact with albumin membranes. Direct cytotoxicity was measured by BSA / NaBr400. [ka] , BSA / NaBr664 [ka] and BSA / CaCl2 membrane [ka] (M / S=25mg / cm 2 The normalized metabolic activity of Balbc 3T3 cells cultured for 24 hours in contact with Ctl+ was estimated by comparing it with the normalized metabolic activity of a positive control (Ctl+). [ka] No significant differences were observed between the groups treated. Biological replicates = 4, total technical replicates = 20. [Figure 12A] 1 shows a microscopic examination of Balbc 3T3 mouse fibroblasts cultured in contact with a BSA / NaBr membrane for 24 hours. Fibroblasts were found surrounding the biomaterial. [Figure 12B] 1 shows a microscopic examination of Balbc 3T3 mouse fibroblasts cultured in contact with a BSA / NaBr membrane for 24 hours. Fibroblasts were found on top of the biomaterial. [Figure 13] After removing the medium, freshly transferred albumin membranes (BSA / NaBr400, BSA / NaBr664, and BSA / NaCl2700, respectively) were placed in empty, unused wells. [ka] Figure 1 represents the normalized metabolic activity of Balbc 3T3 fibroblasts measured above. Cell adhesion is estimated by comparing the normalized metabolic activity of Balbc 3T3 cells between the treated and untreated groups. * ) treatment group and positive control (Ctl+, [ka] A significant difference was observed between the two groups (p<0.05). ** ) Significant differences were observed between treatment groups (p<0.05). Biological replicates=3, total technical replicates=12. [Figure 14A]RAW macrophages were cultured in contact with a BSA membrane at 37°C for 48 hours (M / S = 25 mg / cm², from left to right: BSA / NaBr400, BSA / NaBr664, BSA / CaCl2700, BSA / NaBr400 LPS, BSA / NaBr664 LPS, BSA / CaCl2700 LPS). Nitrite concentrations were measured to assess macrophage activation and inflammatory responses. The untreated group (NT) was cultured without a membrane and without LPS. LPS was added to the medium (50 ng / mL) of the LPS-treated control group (TLPS) and the LPS-activated group (LPS) after 24 hours. (*) Significant differences were observed between the treatment and NT groups (p<0.05). (**) Significant differences were observed between the treatment and TLPS groups (p<0.05). Biological replicates = 3, total technical replicates = 12. [Figure 14B] RAW macrophages were cultured in contact with a BSA membrane at 37°C for 48 hours (M / S = 25 mg / cm², from left to right: BSA / NaBr400, BSA / NaBr664, BSA / CaCl2700, BSA / NaBr400 LPS, BSA / NaBr664 LPS, BSA / CaCl2700 LPS). TNF-α concentrations were measured to assess macrophage activation and inflammatory responses. The untreated group (NT) was cultured without a membrane and without LPS. LPS was added to the medium (50 ng / mL) of the LPS-treated control group (TLPS) and the LPS-activated group (LPS) after 24 hours. (*) Significant differences were observed between the treatment and NT groups (p<0.05). (**) Significant differences were observed between the treatment and TLPS groups (p<0.05). Biological replicates = 3, total technical replicates = 12. [Figure 15A] Amplitude sweep tests are shown for hydrated (in water) BSA / NaBr membranes performed at a fixed frequency of 0.5 Hz, strains ranging from 0.01 to 100%, and room temperature. The storage (G´, Pa) and loss (G”, Pa) moduli are expressed as a function of amplitude (strain, %). Three consecutive amplitude sweep tests were performed. [Figure 15B]Amplitude sweep tests are shown for a hydrated (in water) BSA / NaBr membrane performed at a fixed frequency of 0.5 Hz, strains ranging from 0.01 to 100%, and room temperature. The loss modulus (G", Pa) is expressed as a function of amplitude (strain, %) at which the G" modulus reaches a maximum (Payne effect). Three consecutive amplitude sweep tests were performed. [Figure 15C] Amplitude sweep tests were performed on a hydrated (in water) BSA / NaBr film at a fixed frequency of 0.5 Hz, strains ranging from 0.01 to 100%, and room temperature. The storage modulus (G´, Pa) is expressed as time (seconds). Three consecutive amplitude sweep tests were performed. [Figure 16] 1 depicts cross-sectional evaluation of BSA / NaBr films using SEM. [Figure 17] FT-IR spectra of the center of the amide I band for heat-treated (80° C., 72 h) BSA / NaBr material (solid line) and control BSA / NaBr material (dotted line). [Figure 18A] Figure 1 depicts the formation of BSA / NaBr films under controlled vacuum (200, 600, and 800 mbar). Controls were prepared at atmospheric pressure. Visual aspects of the prepared BSA / NaBr films. [Figure 18B] Figure 1 shows the formation of BSA / NaBr films under controlled vacuum (200, 600, and 800 mbar). Controls were prepared at atmospheric pressure. Relative yield (%, white), water absorption (%, speckled), and initial swelling (%, hatched) of the prepared BSA / NaBr films. [Figure 19] Figure 1 depicts an investigation of the effect of organic solvents (ethanol (%, v / v), DMSO (%, v / v), acetonitrile (%, v / v), and dichloromethane (%, v / v)) on BSA / NaBr solutions before evaporation at 37 °C on the relative yield (%, white), water absorption (%, speckled), and initial swelling (%, speckled) of BSA / NaBr films. A control batch was prepared with 0% solvent (volume ratio solvent / solution). [Figure 20]The relative yield (%, white), water absorption (%, speckled), and initial swelling (%, speckled) of albumin membranes prepared with various combinations of CaCl2 and NaBr salts are shown. The molar ratio of CaCl2 / BSA was set at 400, and the molar ratio of NaBr / BSA was varied from 100 to 1000. A control was prepared with BSA / CaCl2 only. [Figure 21] 1 depicts the preloading of doxorubicin (DOX) in Albupad material (i.e., biomaterial according to the present invention). [Figure 22] Figure 1 shows CLSM images of BSA / NaBr and BSA / CaCl membranes preloaded with various amounts of doxorubicin (DOX) ranging from 0.25 to 1 mg / membrane (membrane mass approximately 400 mg, thickness approximately 500 µm). An unloaded membrane (0) was used as a control. The arrow points to the bottom of the sample. [Figure 23A] Quantification of doxorubicin (DOX) removed during the rinsing process from BSA / NaBr (black) and BSA / CaCl (white) membranes as a function of the initial DOX mass / membrane (μg) for 0, 250, 500, 750, and 1000. Mass of DOX (μg) removed in the rinse solution. [Figure 23B] Quantification of doxorubicin (DOX) removed during the rinsing process from BSA / NaBr (black) and BSA / CaCl (white) membranes as a function of initial DOX mass / membrane (μg) of 0, 250, 500, 750, and 1000. Percentage of DOX removed during the rinsing process. [Figure 24A] Figure 1 shows doxorubicin (DOX) release from BSA / NaBr (μg) films after 35 days in water at 37°C. Films (400 mg) loaded with 0.25 mg (crosses), 0.5 mg (triangles), 0.75 mg (circles), and 1 mg (solid line) of DOX were initially tested. [Figure 24B] Figure 1 shows doxorubicin (DOX) release from BSA / NaBr (%) membranes after 35 days in water at 37°C. Membranes (400 mg) loaded with 0.25 mg (crosses), 0.5 mg (triangles), 0.75 mg (circles), and 1 mg (solid line) of DOX were initially tested. [Figure 24C] Figure 1 depicts doxorubicin (DOX) release from BSA / CaCl (μg) films after 35 days in water at 37°C. Films (400 mg) loaded with 0.25 mg (crosses), 0.5 mg (triangles), 0.75 mg (circles), and 1 mg (solid line) of DOX were initially tested. [Figure 24D] Figure 1 shows doxorubicin (DOX) release from BSA / CaCl (%) membranes after 35 days in water at 37°C. Membranes (400 mg) loaded with 0.25 mg (crosses), 0.5 mg (triangles), 0.75 mg (circles), and 1 mg (solid line) of DOX were initially tested. [Figure 25] Figure 1 shows CLSM images of BSA / NaBr and BSA / CaCl membranes (membrane mass ≈ 400 mg, thickness ≈ 500 μm) preloaded with 0.25 mg of FITC insulin (INS-FITC). An unloaded membrane (0) was used as a control. The arrow points to the bottom of the sample. [Figure 26A] Figure 1 represents the quantification of FITC-insulin (INS-FITC) during the rinsing process (μg) and after 35 days in water at 37°C. Membranes (400 mg) were initially loaded with 0.25 mg of INS-FITC. INS-FITC release. An unloaded membrane (0) was used as a control. [Figure 26B] Figure 1 represents the quantification of FITC-insulin (INS-FITC) during the rinsing process (%) and after 35 days in water at 37°C. Membranes (400 mg) were initially loaded with 0.25 mg of INS-FITC. INS-FITC release. An unloaded membrane (0) was used as a control. [Figure 26C] Figure 1 shows quantification of FITC-insulin (INS-FITC) release (%) from BSA / NaBr (black) and BSA / CaCl (white) membranes after 35 days in water at 37°C. Membranes (400 mg) were initially loaded with 0.25 mg of INS-FITC. INS-FITC release. Unloaded membranes (0) were used as controls. [Figure 27A] Figure 1 represents the visual aspects of grafts prepared for in vivo evaluation: BSA / NaBr, BSA / CaCl2, HSA / NaCl, HSA / CaCl2, and HSA / GLU. [Figure 27B] The water uptake of grafts prepared for in vivo evaluation is shown for BSA / NaBr, BSA / CaCl2, HSA / NaCl, HSA / CaCl2, and HSA / GLU. [Figure 28A] Figure 1 shows the histological evaluation of materials (BSA / NaBr, BSA / CaCl2, HSA / NaBr, HSA / CaCl2, and HSA / GLU) implanted into nude mice after sacrifice. Representative magnification of a histological cut of the implant (marked by an arrow) with surrounding tissue stained with Gomori stain. [Figure 28B] Figure 1 depicts the histological evaluation of materials (BSA / NaBr, BSA / CaCl2, HSA / NaBr, HSA / CaCl2, and HSA / GLU) implanted into nude mice after sacrifice. Representative magnification of a histological cut of the implant (marked by an arrow) with surrounding tissue stained with Gomori and PicroSinius stains. [Example]

[0051] Example 1: Preparation of albumin-based biomaterials produced by evaporation and salt-assisted compression

[0052] material

[0053] Chemical Reagents Bovine serum albumin (fraction V, ≥96%) was purchased from Acros Organics. Human serum albumin (≥96%), ovalbumin (≥98%), and bovine blood-derived gamma globulin (≥99%) were purchased from Sigma-Aldrich.

[0054] Sodium bromide (NaBr), potassium chloride (KCl), and potassium acetate (KC2H3O2) were purchased from Sigma-Aldrich. Sodium chloride (NaCl) was purchased from VWR. Chemicals. Potassium bromide (KBr) was purchased from Acros Organics. Sodium iodide (NaI) and dipotassium phosphate (K2HPO4) were obtained from Prolabo. Potassium iodide (KI) was purchased from Carbo Erba Reagents. Magnesium chloride (MgCl2, anhydrous) and ammonium formate (NH4HCO2) were purchased from Fluka. Calcium chloride (CaCl2, 2H2O) was purchased from Merck. Potassium carbonate (K2CO3) was purchased from Alfa Aesar.

[0055] BCA assay reagents (bicinchoninic acid solution and copper(II) sulfate pentahydrate) were purchased from Sigma-Aldrich. Deuterium oxide (DO) was purchased from Sigma-Aldrich.

[0056] Biological reagents Balbc 3T3 mouse fibroblasts (clone A31 ATCC® CCL-163) were cultured at 37°C, 5% CO2, and 95% humidity in a medium containing stabilized glutamine and sodium pyruvate (Dutscher), 10% (v / v) fetal bovine serum (Dutscher), and 1% (v / v) penicillin-streptomycin solution 100X (Dutscher) (final concentrations: 0.06 mg / mL and 0.1 mg / mL, respectively). Cells were cultured in supplemented Dulbecco's modified Eagle's medium, high glucose (DMEM). Cells were harvested using trypsin (0.5 g / L)-EDTA (0.2 g / L) (Dutscher) for 5 min at 37°C. Thiazolyl blue tetrazolium bromide (MTT) was purchased from Sigma-Aldrich. CellTiter Glo viability assay was purchased from Promega.

[0057] RAW264.7 mouse macrophages (ATCC® TIB-71) were cultured in Dulbecco's modified Eagle's medium-high glucose (DMEM) containing stabilized glutamine (Sigma-Aldrich) and supplemented with 5% (v / v) heat-inactivated fetal bovine serum (Gibco), penicillin (100 U / mL) (Sigma-Aldrich), and streptomycin (0.1 mg / mL) (Sigma-Aldrich) at 37°C, 5% CO2, and 95% humidity. Cells were harvested using trypsin (0.5 g / L)-EDTA (0.2 g / L) (Sigma-Aldrich) for 5 min at 37°C. Escherichia coli (K12) lipopolysaccharide (LPS) was purchased from Invivogen. Purified anti-mouse TNF-α antibody clone 1F3F3D4 and biotinylated anti-mouse TNF-α antibody clone XT3 / XT22 for ELISA testing were purchased from eBioscience / ThermoFisher Scientific. Horseradish peroxidase avidin (avidin-HRP) was purchased from Jackson. P-aminobenzenesulfonamide and acetic acid were purchased from Sigma. N-(1-naphthyl)ethylenediamine dihydrochloride was purchased from Acros Organics.

[0058] method

[0059] Preparation (General Procedure) A solution of BSA (100 mg / mL) and NaBr 1 M (molar ratio NaBr / BSA = 664) was prepared in sodium acetate buffer (0.2 M) at pH 6. This solution was poured into a mold (the bottom of which was covered with a non-stick silicone disk) and allowed to evaporate at 37 °C for 7 days. The resulting dried biomaterial was washed to remove salts and immersed in distilled water at room temperature for 48 hours. The water-insoluble film (BSA / NaBr 664) was then recovered and characterized.

[0060] Initial characterization The salt / albumin molar ratio (Equation 1) and M / S ratio (Equation 2) were used to label the formulations. The relative yield (Equation 3), water absorption (Equation 4), and initial swelling (Equation 5) were used to compare the prepared membranes. The density of the material (Equation 6) was evaluated by immersing the material in distilled water at room temperature.BSA represents the initial weight of albumin used in the formulation. i represents the area of ​​the container used during the evaporation process. W d V represents the weight of the final dry membrane after washing with distilled water for 48 hours and drying overnight in an oven at 37 °C. d is the volume of the dry film measured by immersing the material in distilled water at room temperature. h represents the weight of the hydrated membrane at equilibrium after immersion in distilled water for 24 hours and removal of excess surface water using filter paper. A h is the area of ​​the surface of the hydrated film, calculated after measuring the diameter using an electronic digital caliper (TACKLIFE-DC01, accuracy ±0.2 mm).

[0061]

number

[0062] Standardized Induced Potential (SIP) Measurement A titration solution was prepared by dissolving BSA at a concentration of 1 mg / mL in mQ water. A titration solution was prepared by dissolving NaBr at a concentration of 60 mg / mL in mQ water. The NaBr solution was then used to titrate the protein surface charge by measuring the induced potential using streaming current detection. A Muetek PCD 02 detector was used. 10 mL of BSA solution was transferred to the detector tank. After a 5-minute equilibration period, continuous addition of saline NaBr solution was performed at a frequency of 30 μl / min. The assay was stopped when the measured potential reached a plateau.

[0063] BCA assay BSA / NaBr664 membrane (initial BSA concentration=200mg / mL, M / S=105mg / cm 2, n=3) were washed with 4.5 mL of ultrapure water (2 × 1.5 mL (2 × 30 min), then 1 × 15 mL (2 h)). The volume of each rinse was then adjusted to 5 mL using a volumetric flask. The albumin concentration of the initial solution used for the formulation and the rinse solution was then measured using a BCA test with a standard range (20 μg / mL–1000 μg / mL). The assay was performed in a 96-well plate. The reagent (bicinchoninic acid / CuSO4) was added to the solution (25 μL of protein solution from 200 μL of reagent). The plate was then incubated at 37°C for 30 minutes. Absorbance readings at 560 nm were taken at room temperature using a SAFAS Xenius XM spectrofluorometer (SAFAS Monaco). After calculating the amount of albumin in the membrane, the amount of NaBr was estimated.

[0064] Microanalysis Electron-induced X-ray microanalysis was performed on a Quanta 250 ESEM (FEI) operating at an accelerating electron voltage of 10 kV (emergence angle = 35°, acquisition time = 100 s, processing time = 7.68 μs). A 1000-milliliter (M / S) filter was used (Company, Eindhoven, The Netherlands) to measure the surface area of ​​the sample. Four BSA / NaBr membranes were analyzed: BSA / NaBr400, BSA / NaBr664, BSA / NaBr700, and BSA / NaBr1400 (M / S = 113 mg / cm). 2 The amount of NaBr was estimated using the atomic percentage of sodium bromide in the sample.

[0065] Rheological behavior and compression assay Rheological characterization and compression assays were performed using a Malvern Kinexus Ultra+ rheometer equipped with a 2 cm diameter planar moving device. Hydrated BSA / NaBr664 membranes (M / S = 105 mg / cm) were used. 2, thickness = 1.7 mm) was used. For the vibration protocol, the samples were subjected to a controlled shear stress of 1 Pa, and measurements were performed following a vibration frequency ramp ranging from 100 Hz to 0.01 Hz at 25 °C. For the compression assay, a force ramp from 0.5 N to 40 N (0.04 mm / s) was applied to the samples at 25 °C. The elastic modulus (E) of each sample was calculated within the elastic region of the strain (ε)-stress (σ) curve (Equation 7). σ=E×ε (7)

[0066] Traction assay Traction assays were performed using an Instron ElectroPuls E3000 equipped with a 100 N force sensor. Six hydrated BSA / NaBr664 membranes (M / S ratio 110.9 mg / cm) were used. 2 A batch of 1000 sieves was used. The samples were cut with a punch to form six specimens with standardized dimensions (initial effective length L = 40 mm, effective initial width l = 10 mm, initial thickness e = 1.74 ± 0.079 mm). Tensile tests were then carried out at room temperature with a pulling rate of 0.1 mm / s. The elastic modulus (E) was calculated within the elastic region of the strain (ε)-stress (σ) curve (Equation 7).

[0067] Stability in aqueous solution BSA / NaBr664 membrane (M / S=105mg / cm 2 The membranes were placed in three batches into 25 mL of the following dissolution media: distilled water, saline (0.9% NaCl), acidic medium (pH 3, color indicator: bromothymol blue), basic medium (pH 10, color indicator: bromothymol blue), saline with 1 M NaCl, saline with 1 M NaBr, ethanol, and trypsin solution (0.125 mg / mL diluted in PBS buffer). The media containing the membranes were then incubated at 37 °C with shaking (180 rpm) for 7 days. The membranes were then washed with water and their mass loss was characterized (Equation 8).

[0068]

number

[0069] IR analysis A VERTEX 70 FTIR spectrometer (Bruker, Germany) equipped with a deuterated triglycine sulfate detector (RTDLaTGS) and a KBr beam splitter was used for infrared measurements. The IR spectrum was obtained at approximately 1650 cm using a DO solution. -1 The spectral overlap between the amide I band of water and the strong absorption bands of the water was avoided. All samples were placed between two CaF2 windows. FTIR spectra of the samples were recorded at room temperature between 4000 and 800 cm. -1 , nominal resolution 2cm -1 Spectra were recorded at 10 kHz, accumulating 128 scans per spectrum, with a scan rate of 10 kHz and a D2O spectrum used as background. Liquid samples (BSA, BSA / NaBr400, and BSA / NaBr664 solutions) were prepared in D2O at a BSA concentration of 100 mg / mL. Solid samples (BSA / NaBr400 and BSA / NaBr664 films) were hydrated with D2O.

[0070] Spectral analysis was performed using the spectrometer software OPUS7.5 (Bruker, Germany). For secondary structure analysis, the amide I region (1700–1600 cm) of the deconvoluted spectrum was analyzed. -1 ) was performed. Before curve fitting, low wavenumber (1600 cm -1 ) side and high wave number (1700cm -1The spectra of the amide I bands were baseline-corrected using the minimum value of the α-side. Deconvolution was performed according to a least-squares iterative curve-fitting program (Levenberg-Marquardt) using Gaussian line shapes. The number of subbands and their positions were determined from the deconvoluted spectrum and the second and fourth derivatives of the spectrum. In the final fit, the height, width, and position of all bands were adjusted to reduce the residual RMS error as much as possible (below 0.005), while keeping at least one of these parameters unchanged each time. Finally, the accuracy of the curve fit was confirmed by comparing the second derivatives of the original and fitted curves. The fractional areas of the fitted components were used to calculate the percentages of various secondary structure elements (α-helix, β-sheet, β-turn, and random coil).

[0071] SAXS ICS Small-angle and wide-angle X-ray scattering (SAXS and WAXS) analyses were performed on three controls: a dry BSA / NaBr664 film, a lyophilized BSA powder, and two solutions of BSA (Solution 1: 113.69 mg BSA + 36.31 mg HO, Solution 2: 119.39 mg BSA + 79.61 mg HO). Measurements were performed on a Rigaku diffractometer attached to a microfocus rotating anode generator (Micromax-007HF) operated at 40 kV and 30 mA. The beam was monochromated (wavelength λ = 1.54 Å) and focused with a confocal Max-FluxOptics (Osmics, Inc.) and a three-pinhole collimation system. The scattering intensity was measured as a function of the magnitude of the scattering vector q (Equation 9), where θ is the scattering angle. Two different configurations were used to cover a large scattering vector range.

[0072] The low q range was investigated with a 2D multiwire detector placed at d = 0.81 m from the sample position (0.01 Å -1 <q<0.33Å -1 ). Higher q values ​​were measured using a Fuji imaging plate inserted close to the sample (d = 0.1 m, 0.1 Å). -1 <q<3Å-1 ) Scattering patterns were processed according to the usual procedure for isotropic scattering. Intensities were radially integrated and corrected for electron background, detector efficiency, sample transmittance, and sample thickness. For BSA solutions, scattering from the pure solvent and container was also measured and subtracted. Intensities were converted to an absolute scale using a calibrated lupolen standard. The scattering vector was calibrated using the diffraction peak of silver behenate powder.

[0073]

number

[0074] Scanning electron microscope (SEM) Scanning electron microscope evaluation was performed using a Quanta 250 operating at an accelerating electron voltage of 10 kV. The results were obtained using an ESEM (FEI Company, Eidhoven, The Netherlands) on a BSA / NaBr664 membrane (M / S ratio = 105 mg / cm). 2 The samples were dried at 37°C for 48 hours. The samples were then coated with gold-palladium alloy using a Hummer Jr. sputtering system (Technics, Union City, CA, USA). The surfaces and cross sections were examined.

[0075] Cytotoxicity test of the extract For indirect cytotoxicity assessment, the ISO standard (ISO 10993-5 (2009)) was followed. The membranes used during this test were BSA / NaBr400, BSA / NaBr664, and BSA / CaCl2700. The membranes were coated with 25 mg / cm2 of BSA / NaBr400, BSA / NaBr664, and BSA / CaCl2700. 2The membranes were washed with 70% ethanol, then with sterile PBS 1X, and sterilized under UV light for 15 minutes. They were then stored in sterile PBS 1X until further use. Each membrane was transferred to a 12-well plate and extracted with 1.5 mL of medium (DMEM + FBS (10%) + PS (1%)) for 72 hours at 37°C with stirring. Next, dilutions containing 12.5%, 25%, 50%, and 100% (v / v) extract were prepared. Balbc 3T3 mouse fibroblasts (clone A31 ATCC® CCL-163) were cultured in a 96-well plate at 8000 cells per well (medium: DMEM + FBS (10%) + PS (1%)) for 24 hours at 37°C. The next day, the medium in each well was replaced with 100 μL of diluted extract. Positive and negative controls were prepared with medium alone and medium containing 20% ​​DMSO, respectively. The plates were then incubated at 37°C for 24 hours. After incubation, the medium in each well was replaced with 100 μL of MTT diluted in fresh medium (1 mg / mL), and the plates were incubated at 37°C for 2 hours. The formazan crystals were then solubilized in 80 μL of DMSO and equilibrated at room temperature for 15 minutes. The absorbance at 560 nm was then measured using a SAFAS instrument. The metabolic activity of a positive control was used to determine the percentage of viable cells in each group.

[0076] Direct Cytotoxicity Test The membranes used during this study were BSA / NaBr400, BSA / NaBr664, and BSA / CaCl2700. The membranes were applied in a non-stick silicone mold at 25 mg / cm 2The hydrated membranes were prepared at an M / S ratio of 0.7 mm. The hydrated membranes were cut using a circular punch to obtain small disks (diameter = 5 mm, thickness = 0.7 mm). The disks were washed with 70% ethanol, then with sterile PBS 1X, and sterilized under UV light for 15 minutes. They were then stored in sterile PBS 1X until further use. For direct cytotoxicity assessment, the sterilized disks were transferred to a black-walled 96-well plate. Next, Balbc 3T3 mouse fibroblast cells (clone A31 ATCC® CCL-163) were plated directly onto the biomaterial disks at 8,000 cells per well (medium: DMEM + FBS (10%) + PS (1%)). Positive and negative controls were added to medium alone and medium containing 20% ​​DMSO, respectively. The plates were then incubated at 37°C for 24 hours. After incubation, the plates were equilibrated at room temperature for 30 minutes. The medium was then removed. To each well, 50 μL of fresh medium was added, followed by 50 μL of CellTiter-Glo reagent. Bioluminescence was then measured using the SAFAS instrument and the following protocol: The plate was agitated for 2 minutes and then allowed to equilibrate for 10 minutes before measuring bioluminescence. The metabolic activity of a positive control was used to determine the percentage of viable cells in each group.

[0077] Macrophage activation assay The membranes used during this study were BSA / NaBr400, BSA / NaBr664, and BSA / CaCl2700. The membranes were applied in a non-stick silicone mold at 25 mg / cm 2 The hydrated membranes were prepared with an M / S ratio of 0.7 mm. The hydrated membranes were cut using a circular punch to obtain small disks (diameter = 5 mm, thickness = 0.7 mm). The disks were washed with 70% ethanol, then with sterile PBS 1X, and sterilized under UV light for 15 min. They were then used further. The discs were stored in sterile PBS 1X until 24 h of incubation. For the macrophage activation assay, the sterilized discs were transferred to a 96-well plate. RAW264.7 macrophages were then added directly to the plate at 100,000 cells per well (medium: DMEM + FBS (5%) + PS (1%)). After 24 h of incubation at 37 °C, LPS was added to the LPS-treated group to a final concentration of 50 ng / mL in each well. The plates were then incubated for another 24 h at 37 °C. Positive and negative controls were prepared with medium alone and medium containing 50 ng / mL LPS, respectively. Cell morphology was then evaluated microscopically, and NO and TNF-α production was assessed as follows:

[0078] Assessment of NO production The concentration of nitrite in the cell supernatant was evaluated by the Griess test (n = 3). Sixty microliters of Griess reagent (a v / v mixture of 58.1 mM p-aminobenzenesulfonamide in 30% acetic acid and 3.9 mM Mn-(1-naphthyl)ethylenediamine dihydrochloride in 60% acetic acid) was added to 40 μL of the supernatant, and the absorbance at 543 nm was measured and compared with a standard curve of sodium nitrite.

[0079] Assessment of TNF-α production TNF-α concentrations in cell supernatants were assessed by ELISA (n=3) using commercially available reagents according to the manufacturer's instructions. The capture antibody was diluted to 1 μg / mL in 0.05 M pH 9.6 carbonate / bicarbonate buffer and coated overnight at 4°C, followed by incubation in PBS 0.05% Blocking was performed with Tween® 20 1% BSA (1 hour, 37°C). Samples were then diluted with medium and incubated with capture antibody (2 hours, 37°C), followed by the addition of detection antibody diluted to 0.5 μg / mL in PBS 0.05% Tween® 20 1% BSA (1 hour, 37°C). Avidin-HRP was then introduced (45 minutes, 37°C), and development was performed with a solution of 1.25 mM tetramethylbenzidine and 13.05 mM H2O2 in 0.1 M pH 5 citrate buffer. Finally, development was stopped by adding 1 M HCl, and absorbance was measured at 450 nm.

[0080] statistical analysis Data were analyzed using R (version 3.6.1, R Foundation for Statistical Computing, Vienna, Austria). Normality of distribution was determined with the Shapiro-Wilk test. Equality of variances was determined with the F-test. If the data were normally distributed and the sample variances were equal, two means were compared using a two-tailed t-test. If these two conditions did not apply, a Mann-Whitney test was performed instead. Values ​​were considered statistically significant at p<0.05.

[0081] Results and Discussion

[0082] Prescription Parameter Screening The solubility of albumin in aqueous solutions and its thermal and pH stability regions were thoroughly studied. Previous studies have shown that albumin is stable in the pH range of 3 to 9. It has also been shown that the denaturation temperature is pH-dependent and decreases at lower pH values ​​(62°C at pH 7.4 and 46.8°C at pH 3.5). In this work, the evaporation temperature was set at 37°C and the pH at 6 to preserve the native structure of albumin as much as possible. Next, a thorough screening of operating conditions was conducted to identify those that would enable the formation of interesting biomaterials. Albumin solutions (protein only or containing salts) formulated at a controlled pH (pH = 6) were evaporated in an oven at 37°C until the residue was completely dry (see Figure 1). The residues were then washed to remove excess salt and protein, and then soaked in distilled water for 48 hours to evaluate their water solubility. Only formulations that produced water-insoluble materials were selected. This work highlights the potential of the biomaterials field. In terms of potential applications, water-insoluble membranes that exhibit excellent handling properties are the most promising.

[0083] 105mg / cm 2For the material formulated with M / S of 105 mg / cm, a solid material was obtained after 68–69 hours (see Figure 2). After 69 hours, a thin white layer of excess salt formed on the surface of the material as the residual water evaporated. After washing, the salt layer was quickly removed, leaving a translucent film (see Figure 2). To allow for the formation and compaction of the material, an M / S of 105 mg / cm was used. 2 An evaporation period of 7 days was found to be optimal for the films.

[0084] First parameter: salt First, we verified the solubility of albumin residue prepared without salt to confirm the importance of salt in albumin membrane formulation. Next, we performed a thorough screening of numerous salts at three different concentrations (0.5 M, 1 M, and 2 M). As previously described, we used the water solubility of the dried residue after 48 h in distilled water to identify salts that enable membrane formation.

[0085] According to the Hofmeister series, different ions affect protein stability and solubility differently. The lyotropic effect is related to the size, charge density, and polarizability of the ions. When working with solutions containing high concentrations of salt and protein, the effects of interactions between solvent molecules, salt ions, and the protein must be considered. In this experiment, 12 salts with various anion-cation couples were tested: KCl, KBr, KI, NaCl, NaBr, NaI, CaCl2, MgCl2, KC2H3O2, NH4HCO2, K2CO3, and K2HPO4. The results of this experiment suggest that albumin membrane formation depends on both the type of salt and its concentration. In the presence of KCl, NaCl, or KBr, albumin molecules do not organize into membranes, and the dry residue is a mixture of crystallized salt and dry protein, which is completely water-soluble. Furthermore, premature aggregation of albumin in initial solutions prepared with salts containing divalent anions (K2CO3 and K2HPO4) prevents membrane formation. Water-insoluble films were obtained with seven of the 12 salts: NaBr, KI, NaI, CaCl2, MgCl2, KC2H3O2, and NH4HCO2. With NaBr, films were obtained at all three concentrations tested. However, with the other six salts, albumin films were obtained only at certain concentrations. The physical aspects and properties of these films (water absorption, initial swelling, handling) varied significantly depending on the type of salt used and its concentration.

[0086] The BSA / NaBr664 membranes (initial salt concentration = 1 M) showed excellent mechanical strength and handling properties. These membranes (M / S = 105 mg / cm 2 ) were produced with a relative yield of 87.6% ± 4.1%, their water absorption and initial swelling were estimated to be 123.1% ± 6.8% and 121.6% ± 4.7%, respectively (n = 8), and their density was 1.29 ± 0.02 g / cm 3 This formulation was chosen as a reference for identifying the parameters of albumin-based film formation and for characterizing these materials.

[0087] Second parameter: Salt / albumin molar ratio It has previously been shown that salt concentration influences membrane formation. However, it is unclear whether salt concentration should be considered an independent parameter or whether it should be combined with the albumin concentration in a particular solution. The effect of the initial concentrations of BSA and NaBr on membrane formation was evaluated by comparing membranes obtained in two assays. In the first assay, a constant concentration of NaBr and variable concentrations of BSA, and therefore variable salt / albumin molar ratios, were used. In the second assay, however, both concentrations were varied simultaneously without changing the salt / albumin molar ratio. In the first assay, solutions of 100 mg / mL, 200 mg / mL, 300 mg / mL, and 400 mg / mL BSA were prepared in 1 M NaBr. The NaBr / BSA molar ratios were 664, 332, 221, and 166, respectively. Decreasing the molar ratio significantly decreased the yield of the formulation (86.4%, 77.2%, 62.7%, and 0%, respectively), and the resulting films differed significantly in terms of visual aspect and water absorption. The residue obtained after evaporation of the solution with an albumin concentration of 100 mg / mL (molar ratio of salt / albumin = 166) was completely water-soluble. In the second assay, the prepared solutions contained the following concentrations of BSA and NaBr: 100 mg / mL for 1M, 200 mg / mL for 2M, 300 mg / mL for 3M, and 400 mg / mL for 4M. The NaBr / BSA molar ratio was 664 for all solutions. Unlike the first assay, all solutions in the second assay resulted in membrane formation. The resulting membranes shared the same visual aspect and exhibited similar properties. Therefore, the effect of salt concentration on membrane formation cannot be evaluated without combining it with the albumin concentration in the initial solution. Because membrane formation depends on the combined effect of both salt and albumin concentrations, the salt / albumin molar ratio proves to be a more appropriate and reliable parameter for assessing albumin membrane formation.

[0088] The next step was to identify the range of NaBr / BSA molar ratios that could form membranes. BSA solutions were prepared with a set concentration of albumin (100 mg / m) and NaBr / BSA molar ratios of 50–2000. These solutions were evaporated, and the resulting materials were immersed in water for 48 hours as described above. Fully formed membranes were obtained within the molar ratio range of 100–3000, especially within the range of 200–2000. At the lowest and highest molar ratios in this range, the resulting membranes were less robust and more susceptible to breakage and degradation during handling, but this was acceptable.

[0089] Effect of albumin surface charge Due to the well-established implication of the ionic content of the initial solution in the formation of albumin films, it is necessary to evaluate the surface charge of albumin to provide a better understanding of the ionic phenomena that lead to film formation. The surface charge of albumin depends on the pH of the initial solution and its ionic strength.

[0090] To evaluate the effect of pH on membrane formation, albumin and NaBr solutions at a molar ratio of 664 NaBr / BSA were prepared at pH values ​​of 4, 5, 6, 7, and 8. With an isoelectric point of 4.7 and an isoelectric point of 5.2, BSA has a net negative charge at pH 6, 7, and 8, a net positive charge at pH 4, and becomes zwitterionic around pH 5. Albumin membranes were obtained at all pH values ​​tested. These membranes shared similar visual appearance and formulation yields, but their water absorption and initial swelling varied significantly. Membranes formulated at the highest pH value exhibited higher water absorption and initial swelling. Thus, pH appears to have a moderate effect on membrane formation. Furthermore, studies of albumin surface charge at pH 6 revealed that adding salt increases the overall surface charge of the protein due to interactions between the protein and salt cations. Indeed, the measured induced potential increases significantly with increasing salt concentration before reaching a plateau (see Figure 3). An increase in the surface charge of albumin can result in a decrease in the electrostatic repulsion between molecules, promoting their aggregation to form an albumin film.

[0091] Residual salt In anticipation of biological evaluation, the final composition and residual salt content of the formulated material must be fully characterized. Therefore, two complementary methods were used to determine the final composition of the BSA / NaBr664 membranes and quantify residual NaBr. First, the BCA assay was used to quantify albumin in the rinse water used to wash the BSA / NaBr664 membranes. After evaporating the rinse solution, the dried residue was weighed, and the amount of NaBr removed by the washing process was calculated and compared to the amount of NaBr initially used to form the membranes. The residual NaBr content of the washed BSA / NaBr664 membranes was estimated to be less than 1% (wt%). To verify these results and specifically target the bromine content, the final composition of the BSA / NaBr664 membranes was analyzed directly by microanalysis. Microanalysis was performed directly on the dried membranes and revealed that the bromine content of the tested membranes was undetectable in the analyzed area (analyzed area = 1180 μm², sample thickness = 1 mm). Furthermore, the BSA / NaBr664 membranes were analyzed by microanalysis. Analytical X-ray diffraction of the aBr film (see Figure 8) did not detect any traces of crystalline NaBr in the film. In conclusion, the NaBr initially added to the solution for the formulation of the albumin-based biomaterial is eliminated during the washing process.

[0092] mechanical properties The viscoelastic behavior of selected albumin-based biomaterials was studied after saturation with water. The storage modulus (G´) was found to be higher than the loss modulus (G´´) (see Figure 4). Furthermore, no sol-gel transition was observed. Thus, within the tested frequency range, the films exhibit solid-like behavior.

[0093] During compression assays, the elastic modulus of the membrane formulated with a molar ratio of 664 BSA and NaBr was found to be 0.7 MPa. Furthermore, tensile tests were performed on batches (n=6) of albumin membranes formulated in the same way. The calculated elastic modulus of the membrane after traction assays is 0.87 ± 0.12 MPa. Thus, both assays yield similar results. The maximum stress that caused failure of the biomaterial was estimated to be 0.19 ± 0.03 MPa.

[0094] Table 1 below shows a comparison of traction and compression results. The membranes used were formulated with BSA (bovine serum albumin) and NaBr at a molar ratio NaBr / BSA of 664 under selected conditions (evaporation at 37°C and pH=6). Tests were performed on hydrated biomaterials.

[0095] [Table 1]

[0096] Stability in aqueous solution It is important that biomaterials used in contact with biological fluids have good stability in aqueous media. For applications such as the formulation of implants or scaffolds for tissue regeneration, the biomaterial must be insoluble in water or have a very slow degradation process. Therefore, the stability of the membrane BSA / NaBr664 in aqueous solution was tested. The biodegradability of the membrane was also tested in trypsin solution (see Figure 5).

[0097] Membranes incubated in contact with trypsin solution were completely degraded and dissolved in the buffer solution. Each batch of membranes lost less than 10% of their initial mass in each dissolution medium, except for membranes incubated in basic solution, which lost 17% for membranes incubated for 7 days at 37°C without proteases. Furthermore, formulated albumin-based membranes are insoluble in aqueous media and remain intact for more than 7 days of incubation in these media at 37°C. In fact, these membranes can be stored in distilled water for up to 1 month without showing any deterioration. Furthermore, these biomaterials are resistant to both acidic and basic pH. The mass loss observed in water and ethanol was primarily due to the adhesion of membranes to the sides of the tube during agitation. This can be explained by the erosion of the membrane caused by friction against the surface. Therefore, these albumin-based membranes are very stable in aqueous solution and are completely biodegradable.

[0098] We also tested the stability of BSA / NaBr664 membranes in solutions of urea (2 M, 4 M, and 8 M) and 2-mercaptoethanol (0.1 M). Urea and 2-mercaptoethanol are well-known denaturants that can induce protein unfolding by breaking hydrogen bonds and disulfide bridges, respectively. While the albumin-based membranes tested did not break or dissolve, the diameter (Ah) of the hydrated membranes increased significantly, indicating an increase in initial swelling (E%), and the complex modulus (G * ) indicates a decrease in their elastic modulus (E) (see Figure 6). Furthermore, disruption of hydrogen bonds and disulfide bridges altered the structure of the membranes but did not cause their disassembly.

[0099] Assessment of albumin conformation

[0100] IR analysis. Fourier transform infrared spectroscopy (FTIR) is an established method used to evaluate the secondary structure of proteins. The infrared spectrum of a protein is characterized by a series of absorption regions in the absorption spectrum known as the amide region and the C–H region, respectively. Information about secondary structure is primarily found in the amide I region (1700–1600 cm). -1 ) and the amide II region (1600–1500 cm -1 ) spectrum. The amide I region primarily reflects the C=O stretching vibration of peptide groups and provides information about the secondary structure of proteins. Furthermore, this technique allows for the investigation of the secondary structure of proteins contained in highly concentrated solutions or solid materials, and either hydrated or dried forms can be used. FTIR was used to evaluate the structure of albumin in BSA / NaBr films. In this experiment, to prevent the HO band from interfering with the amide I band, which is in the same absorption range, DO was used to hydrate a ready-to-use BSA / NaBr664 film. Two control solutions were prepared: a solution of BSA (100 mg / mL) and a solution of BSA and NaBr comparable to that used to prepare the BSA / NaBr664 film (BSA concentration = 100 mg / mL, BSA / NaBr ratio = 1:664).

[0101] Analysis of the deconvoluted spectrum of the amide I band revealed the presence of several subbands, which were identified using data already available in the scientific literature. Six subbands were found in the amide I band of the tested samples: α-helix (1655 cm), -1 ), β-sheet (1612, 1629, 1678 cm -1 ), β-turn (1669cm -1 ), random coil (1643cm -1 ) subbands (see Figure 7). The percentages of these secondary structures were then calculated (residual RMS error <0.005). Very similar percentages of β-sheet, β-turn, α-helix, and random coil were obtained for albumin in BSA and BSA / NaBr664 solutions. Thus, under established experimental conditions, NaBr does not alter the secondary structure of albumin. However, in BSA / NaBr664 membranes, there appears to be an increase in β-sheet and β-turn and a decrease in α-helix and random coil (Table 2). Protein unfolding is characterized by an increase in random coil, a disorganized secondary structure. In membrane formulations, albumin appears to favor β-organized structures over these few disorganized structures.

[0102] Table 2 below shows the percentage analysis of each secondary structure identified in the amide I band of albumin present in a BSA solution (100 mg / mL in DO), a BSA / NaBr664 solution (BSA concentration = 100 mg / mL in DO), and a BSA / NaBr664 membrane (residual RMS error <0.005). A BSA solution (BSA 100 mg / mL in DO) was incubated overnight at 80°C and used as a standard for denatured protein.

[0103] [Table 2]

[0104] Crystallography SAXS ICS Small-angle and wide-angle X-ray scattering measurements (SAXS, WAXS) investigate interatomic distances within a sample. In the case of proteins in solution, the hierarchical structure levels of the macromolecule are revealed. Thus, distinct scattering vector domains represent quaternary and tertiary structures (molecular shape and size, q < 0.2 Å). -1 ), inter-domain correlation (≈0.2 <q<≒0.5Å -1 ), intra-domain organization (≒0.5 <q<≒0.8Å -1 ) and secondary structure (≈1.1 <q<≒1.9Å -1 )) are linked to the characteristic configuration of molecules. WAXS is very sensitive to structural disturbances and fluctuations and can reveal small changes in macromolecular organization. However, data interpretation is very complex and often requires comparison with theoretical calculations based on crystallographic results. In the dilute regime, where there are no intermolecular correlations, scattering measurements reflect only intramolecular properties (form factors). With increasing concentration or bulk film properties, this is no longer the case and intermolecular correlations contribute profoundly to the scattering intensity.

[0105] The structural organization of BSA / NaBr664 membranes was investigated by SAXS and WAXS. Washed and dried membranes were measured (e, Figure 2.S8B) and compared with native BSA (a, Figure 2.S8B). Two BSA solutions (b and c, Figure 2.S8B) prepared at different concentrations (solution 124.2 wt% HO, solution 240 wt% HO) were also considered as references for the interpretation of scattering patterns. The experimental intensities are shown in Figure 2.S8A (solution 2) and compared with theoretical curves calculated with CRYSOL and based on the crystallographic description of monomeric BSA (PDB atomic coordinates 3v03). The scattering intensity ranges from 0.1 to 1 Å. -1 Between 0.1 Å and 1.0 Å, both the experimental and theoretical curves correspond well. The small deviation certainly reflects the dynamic fluctuations and disorder within the protein in solution. -1 Below 0.084Å -1 A peak was observed around 0.09 Å. This is related to the spatial correlation between BSA molecules arising from electrostatic repulsion, which was not taken into account in the calculation. With increasing protein concentration (solution 1), this peak appeared at a higher q value (0.09 Å) because the distance between the centers of mass of the molecules became shorter. -1) shifts to a high q value (>1Å -1 ) is 1.5Å -1 A large maximum was observed near the q-axis. In this region, the accuracy of the model is low, but this is outside the acceptable q-range of the program (approximately 0.5 Å). -1 ) CRYSOL is therefore able to explain the main characteristics of the data for BSA in solution: since the protein contains mainly α-helices in the crystalline order state (used in the calculations), the α-helix is ​​approximately 0.2 Å. -1 The predicted shoulder observed at (d=2π / q ∼30 Å) is probably related to the correlation between the α domains, but is 0.4–0.7 Å. -1 The three contributions between (9 and 15 Å) are related to the internal structure of the domain, more specifically to α-helical packing. The theoretically inaccurate description of the 1.5 Å -1 A broad maximum (approximately 4 Å) before and after is found in all proteins, making it less sensitive to the main features of internal organization (α-helix or β-sheet). .

[0106] The scattering pattern of lyophilized powdered BSA is shown in Figure 2.S8B(a). This amorphous sample is used as a reference for unsolvented native BSA. The intensity is significantly altered compared to a simple BSA solution. Data interpretation becomes more complex because intra- and intermolecular correlations contribute to the scattering intensity. Disorder, intermolecular correlations, or specific rearrangements could explain potential changes in the scattering curve between the solution and solid states. It is no longer possible to determine the shape and size of the protein; only shorter length scales related to internal structure or intermolecular correlations can be measured. Initially, a large increase is observed at very low q. This behavior is not related to any particular organization and is simply a result of the powder nature of the sample (Porod scattering). In the absence of such contributions, the intensity tends to a constant low value (q → 0). 0.1 → 0.3 Å -1 In the range of 0.19Å -1A small maximum was observed around (33 Å). This peak could be related to the high concentration limit of the correlation peak observed for "globular" BSA molecules in solution. However, this is unlikely because the average distance between contacting proteins (33 Å) is very close to the radius of gyration (Rg = 28.7 ± 1.5 Å). This maximum is more likely related to correlations between protein domains (including intra- and intermolecular contributions) and is closer to the previously observed ~0.2 Å in BSA solutions. -1 (pure intramolecular contribution). Therefore, the α-domain is still present in the solid state. The other changes are 0.3-1 Å. -1 The structured mass of the BSA solution is observed between 0.66 Å. -1 This maximum indicates a single average distance associated with the α-helix of the BSA domain, which has a characteristic packing distance of 9.5 Å (a classical value for this type of organization). Finally, a single contribution of 1.42 Å -1 A significant contribution was still observed near q. Compared to the BSA solution, the width of the maximum narrowed and its position shifted to smaller q values. This observation may be related to the increased β-sheet content of solid BSA, which is characterized by an interstrand distance of 4.7 Å. Thus, the scattering pattern of powdered BSA indicates a slight change in the internal structure in the disordered solid state.

[0107] The scattering curve of the BSA / NaBr664 film is shown in Figure 2.S8B(e). -1 The increase observed below is related to the porosity of the film (Porod scattering). No Bragg peaks appear, indicating the absence of crystalline NaBr in the film (the pure NaBr salt diffractogram is shown in Figure 2.S8B(d) for comparison). Compared to pure amorphous BSA powder, only small but significant changes were observed. The first peak, related to interdomain correlations, appears at a lower q value (0.19 Å). -1 ~0.17Å -1), indicating large distances between protein domains (33.1 Å to 37.9 Å) (internal and mutual contributions). This behavior is difficult to interpret but has been attributed to subtle changes in tertiary structure, such as the contraction of the α-domain. 0.65 Å associated with α-helical packing. -1 The peak around 1.4 Å is still present but is less intense than in pure BSA, consistent with a change in the helical organization of the membrane compared to BSA powder. -1 The shape of the large maximum near 1.37 Å has also changed. -1 The α / β structure ratio of BSA is slightly altered in the material prepared. These findings support the results obtained by FTIR in the analysis of the amide I band.

[0108] SEM surface analysis To study the surface topography of membranes made with NaBr (molar ratio NaBr / BSA = 664) and evaluate their porosity, the membranes were analyzed using SEM. The surface of the membrane appears very rough with many pores and pore-like substructures (A, Figure 9). Observation of a slice (cross section) of this same membrane reveals the presence of small pores inside the biomaterial, although they appear less numerous than one would expect from observing the surface (B, Figure 9). Surface The unevenness of the is mainly due to the evaporation used in the formulation.

[0109] Other albumin proteins The developed formulation procedure produces albumin-based membranes using BSA with high reproducibility. To study the feasibility of these membranes, formulations were performed with other albumin proteins. Two albumins were selected: human serum albumin (hSA), which has a structure very similar to bovine serum albumin, and ovalbumin (OVA), which differs in structure and molecular weight from the other two proteins. Interesting membranes were obtained with both hSA and OVA. These have morphologies different from BSA membranes. Furthermore, they are more easily hydrated, resulting in higher water content and initial swelling. In conclusion, the established formulation process can be used to prepare albumin-based membranes, regardless of the protein's origin. However, due to the low cost of this protein and its close similarity to the human counterpart, only BSA-based membranes were further characterized in this study.

[0110] Other membrane formulations were tested with different proteins, such as γ-globulin. The main limitation is the protein's solubility in water. Indeed, albumin has an unparalleled solubility in water, allowing the preparation of very highly concentrated solutions. If the protein concentration needs to be reduced due to the protein's solubility threshold, the amount required to reach a minimum thickness compatible with membrane handling must be increased, significantly prolonging the evaporation process. γ-globulin membranes (water solubility at 20 °C ≈ 20 mg / mL) were successfully formulated by evaporating a solution of bovine γ-globulin and NaBr (molar ratio salt / protein = 664, M / S = 35 mg / cm). 2 ). Thus, the formulation procedure developed in this work proves promising for the formulation of protein-based membranes.

[0111] Biological assays

[0112] Cytotoxicity and cell adhesion The biological properties of BSA / NaBr664 membranes were studied. To provide a basis for comparison, two other interesting membranes, BSA / NaBr400 and BSA / CaCl2700, were included in these experiments. First, the cytotoxicity of membrane leachable components was assessed by incubating Balbc3T3 mouse fibroblast cells in the membrane extract. For this cell line, cell viability can be estimated by measuring metabolic activity. Next, the normalized metabolic activity of cells cultured with each extract was compared with that of untreated cells (positive control). In this experiment, the extracts were diluted to reveal a dose-dependent effect. Statistical analysis of the resulting data concluded that there was no significant difference in metabolic activity between the untreated group and groups treated with various dilutions of BSA / NaBr membrane extract (see Figure 10). Furthermore, as previously shown, the non-cytotoxicity of albumin and the absence of NaBr in the final membrane are consistent with the observed non-cytotoxicity of the leachable components of the BSA / NaBr membrane. Next, direct cytotoxicity was assessed by directly incubating Balbc 3T3 cells with these membranes. In this experiment, relatively thin membranes (average thickness approximately 0.7 mm) were used, allowing for microscopic tracking of cell-material interactions (see Figure 11). Statistical analysis of the data revealed no significant differences in metabolic activity between untreated cells and cells cultured on the tested membranes (see Figure 10). Furthermore, microscopic examination revealed that fibroblasts were spreading around and on top of the membranes (see Figure 12). To quantify cell attachment to the membranes, Balbc 3T3 cells were incubated with the membranes for 24 hours. The medium was then removed, the membranes were transferred to empty wells, and the metabolic activity of the cells on each membrane was measured. Significant differences were observed between the BSA / NaBr664, BSA / NaBr400, and BSA / CaCl2700 groups (p<0.05). Approximately 45% (44.55% ± 10.67%) of the seeded fibroblasts attached to the BSA / NaBr664 membrane. The estimated percentage was lower for the BSA / NaBr400 membrane (27.36% ± 11.22%) and higher for the BSA / CaCl2700 membrane (77.62% ± 20.26%). (See Figure 13). Therefore, the initial formulation solution appears to have a significant impact on cell-membrane interaction, even though salts are completely removed during the washing process. In conclusion, the formulated albumin-based biomaterial is non-cytotoxic and favors cell adhesion and colony formation.

[0113] Macrophage activation The effect of albumin membranes on macrophage activation was evaluated by measuring the concentrations of nitrite and TNF-α. NO and TNF-α are produced by activated macrophages to initiate and maintain inflammatory responses. Live macrophages were cultured with the tested membranes for 24 hours. LPS was then directly introduced into the wells of the LPS-activated group to activate the macrophages, and the cells were incubated for another 24 hours. Compared to the untreated group (NT), nitrite production slightly increased in the presence of the tested membranes (p<0.05). LPS activation caused a significant increase in nitrite concentration in the medium. However, nitrite production appeared to be significantly reduced when macrophages were cultured with albumin membranes compared to the LPS-treated control (TLPS) (p<0.05). TNF-α production followed a similar trend to the inactivated group. However, there was no significant difference in TNF-α production between the BSA / NaBr group and the T LPS group, which differed from the BSA / CaCl2700 group, which showed a significant decrease in TNF-α production (p<0.05). Therefore, the albumin-based membranes tested do not efficiently induce inflammatory responses by activating macrophages.

[0114] conclusion In these studies, several interesting biomaterial models were developed using salts such as NaBr, NaI, KI, CaCl2, MgCl2, potassium acetate, and ammonium formate. The properties of these membranes can be tuned by changing the formulation parameters. Two key parameters were identified: the presence of salt and the molar ratio of salt to albumin. Furthermore, it was possible to obtain membranes with ternary systems such as salt 1 / salt 2 / albumin or salt / albumin / polymer. These systems allow for the tuning of membrane properties (mechanical and intrinsic) and the acquisition of functionalized biomaterials with novel properties. Albumin-based biomaterials prepared by evaporation in the presence of salts form versatile models whose properties can be adjusted to suit the requirements of the targeted therapeutic application.

[0115] Example 2: Physicochemical investigation and evaluation of the versatility of the technology, loading of active substances, and preliminary evaluation of biocompatibility and in vivo biodegradability

[0116] 2.1 Physicochemical investigation and evaluation of the diversity of the technology In the following experiments, BSA / NaBr materials were prepared by evaporating a solution of BSA (initial concentration = 100 mg / mL) and NaBr (initial concentration = 62 mg / mL) in sodium acetate buffer (pH = 6) at 37 °C until a dry material was formed. BSA / CaCl materials were prepared by evaporating a solution of BSA (initial concentration = 100 mg / mL) and CaCl (initial concentration = 155 mg / mL) in sodium acetate buffer (pH = 6) at 37 °C until a dry material was formed.

[0117] Stability in organic solvents. Organic solvents, where the majority of active substances are lipophilic molecules, are very conducive to solubilization and loading into biomaterials. Furthermore, loading (postloading) of drugs into easily formed materials requires the stability of the material in the solvent used to solubilize the drug. The stability of Albupad material (i.e., biomaterial according to the present invention) in organic solvents was evaluated in the following solvents: ethanol, DMSO, acetonitrile, and dichloromethane. BSA / Na Br and BSA / CaCl2 membranes were placed in each solvent at room temperature for 72 hours. Their stability was assessed by comparing their mass loss. After 72 hours of incubation, the membranes showed no mass loss in all solvents tested. Furthermore, their physical properties showed no visible signs of deterioration, and their hydration properties were maintained. Thus, Albupad materials are stable in ethanol, DMSO, acetonitrile, and dichloromethane.

[0118] Rheological evaluation of BSA / NaBr films. In this study, amplitude sweep tests were performed on hydrated (in water) BSA / NaBr films (n = 4) to study their viscoelastic behavior. G' represents the elastic or recoverable component, and G'' is the viscous component. During each test, the frequency was set at 0.5 Hz, and the strain was increased from 0.01 to 100%. None of the tested samples showed any noticeable damage, and a strong slip effect was observed above 10% strain, rendering the data unusable (omitted). In the linear viscoelastic region (LVE) below 1% strain, G' (59.6 ± 5.1 kPa) is higher than G'' (7.7 ± 0.8 kPa) (Figure 15A). Therefore, in this range, the material behaves as a solid elastic material. After 1% strain, G'' increases, reaching a maximum at a strain of 1.79 ± 0.07%, where the Payne effect is identified (Figure 15B). This effect is characteristic of materials composed of two phases, with the matrix containing the harder particles suspended, causing greater energy dissipation at a given deformation. This effect has been primarily described in rubber elastomers filled with carbon black particles and is attributed to deformation-induced changes in the material's microstructure. Thus, the BSA / NaBr material is composed of a matrix containing harder particles. However, it was determined that the salt was removed during the washing process. Therefore, the particles are likely albumin aggregates / particles surrounded by a softer albumin matrix. This result was confirmed by MEB analysis of sections of the BSA / NaBr material, revealing the particle structure that forms these materials (Figure 16). When the material was subjected to multiple successive amplitude sweep tests (Figure 15C), G' recovered its initial value, proving that the cohesion of the material did not change during the experiment. Similar observations were made in several other formulations, such as BSA / CaCl2 membranes, indicating that these rheological findings are specific to Albupad materials.

[0119] Contact angle of BSA / NaBr films. Contact angle measurements were performed by depositing a microdrop of milliQ water (5 μL) onto the surface of a dried sample of BSA / NaBr film (deposition rate = 2 μL / s). Images were recorded for 100 seconds (1.4 fps). During these tests, contact angles varying from 90° to 100° were measured. The kinetics of film hydration and droplet sinking into the material were slow (>30 min) and therefore could not be observed during these measurements. Therefore, the surface of the dried BSA / NaBr film is moderately hydrophilic due to its slow hydration rate and high surface roughness (as observed by SEM analysis in previous tests).

[0120] Accelerated degradation of BSA / NaBr membranes. Dried BSA / NaBr membranes were placed in an oven at 80°C for 3 days. The amide I spectra of the heat-treated membranes were then analyzed using FT-IR and compared with those of the control membrane. The amide I band of the heat-treated membrane was similar to that of the untreated control (Figure 17). Therefore, the secondary structure of albumin within the membrane was not altered by heating under the test conditions.

[0121] Fabrication under controlled vacuum. Fabrication of Albupad materials by evaporation under vacuum can provide a useful tool for reducing the time required for evaporation. Therefore, the feasibility of formulation under controlled pressure was carried out using a vacuum oven. A solution of BSA / NaBr (initial concentration of BSA = 100 mg / mL, initial concentration of NaBr = 1 M) was evaporated in a vacuum oven at 37 °C for 24 h, and the following pressure values ​​were tested: 800, 600, and 200 mbar. The dried materials were then washed and immersed in water for 48 h to evaluate their stability. Furthermore, physical appearance, relative yield, water absorption, and initial swelling values ​​were used. The materials formed under controlled vacuum were characterized and compared with a control batch prepared under atmospheric pressure. After evaporation of the BSA / NaBr solution under controlled vacuum at all pressure values ​​tested, handleable, water-insoluble materials were obtained. The formed membranes had similar physical characteristics and handling properties to the control batch. However, removal of soluble gases from the solution during membrane formation resulted in trapped air bubbles in the membrane structure, leading to the formation of large pores at 200 and 600 mbar (Figure 18A). Therefore, formulation optimization is necessary to control or prevent the formation of these pores, for example, by adding a step to degas the solution before evaporation. Furthermore, the relative yield of the formulation under vacuum (60%–70%) was relatively lower than that of the control batch (>90%) (Figure 18B). Next, the mechanical and rheological properties of the membranes prepared at 600 mbar were evaluated. Compression assays were performed on hydrated BSA / NaBr membranes (in water) at 600 mbar using a rheometer with a parallel plate measurement system. The membrane's modulus was 0.76 MPa, similar to that of the control batch and previously measured for BSA / NaBr materials. Amplitude sweep tests were performed on hydrated (underwater) BSA / NaBr membranes (n = 4) to study their rheological properties. During each test, the frequency was set at 0.5 Hz, and the strain was increased from 0.01 to 100%. These experiments revealed that BSA / NaBr membranes compounded under a controlled vacuum of 600 mbar possessed similar rheological properties to BSA / NaBr membranes compounded under atmospheric pressure. Therefore, preparation under a controlled vacuum allows for the formation of albumin pad materials without altering their properties.

[0122] Solvent Investigation. The use of organic solvents, such as ethanol, DMSO, and acetonitrile, offers an interesting tool for incorporating poorly water-soluble active substances into albumin and salt solutions, thereby providing a method for preloading these molecules into Albupad material. The organic solvent is easily removed during evaporation (in the case of volatile solvents such as ethanol) or washing (in the case of water-miscible solvents such as DMSO). Therefore, a feasibility study of formulating Albupad material in a mixed organic solvent / albumin solution was tested. Four solvents were selected for this investigation: ethanol, DMSO, acetonitrile, and dichloromethane. The solvents were added directly to the BSA / NaBr solution according to solvent / solution volume ratios of 2.5, 5, 10, 15, 20, 25, and 30% v / v. The solvent / solution mixture was then allowed to evaporate at 37°C for 7 days. The dried material was subsequently washed with distilled water for 48 hours. The relative yield, water absorption, and initial swelling of the formulated material were compared to a control batch formulated without organic solvent (volume ratio = 0%). Stable, water-insoluble films were formed in the presence of all four solvents at all ratios tested. These materials were easy to handle and shared similar physical characteristics to the control batch. For materials formulated in the presence of ethanol, films prepared at volume ratios ranging from 2.5% to 15% had similar relative yields (85-88%), water absorption (238-250%), and initial swelling (137-145%) compared to the control batch values ​​(Figure 19). Films prepared at volume ratios ranging from 20% to 30% showed a decrease in relative yield (80-82%) and an increase in water absorption (304-346%) and initial swelling (147-154%) compared to the control batch (Figure 19). The presence of DMSO did not affect the relative yield of the formulation or the initial swelling values ​​of the materials. The water absorption values ​​of membranes prepared at volume ratios ranging from 2.5% to 10% were comparable to those of the control batch, whereas those prepared at volume ratios ranging from 15% to 30% were higher (194-345%) (Figure 19). Regarding the tested water-immiscible solvents (acetonitrile, dichloromethane), all prepared membranes shared similar relative yields, water absorption rates, and initial swelling values ​​(Figure 19). In conclusion, the presence of up to 30% (v / v) organic solvent does not interfere with membrane formation and allows for the preparation of handleable materials.No changes in the properties of these materials were observed compared to control batches containing up to 15% ethanol, 10% DMSO, and 30% acetonitrile or dichloromethane. Similar results were obtained for BSA / CaCl membranes, and BSA / NaBr and BSA / CaCl sponges.

[0123] Salt Combinations. Salt is a key parameter for formulating albumin materials using Albupad technology, and the type of salt and its concentration are suitable tools for adjusting the material properties. Furthermore, salt combinations provide an additional tool for better tuning of material properties, allowing the Albupad material platform to adapt more flexibly depending on potential application requirements. Therefore, the applicability of Albupad technology was investigated for formulating albumin materials using salt combinations. In these experiments, various combinations of two different salts were tested: primary salt (S1) and secondary salt (S2). The salt / albumin molar ratio for S1 was set, and the salt / albumin molar ratio for S2 was varied from 100 to 1000. The following salt combinations were tested: NaBr400 / CaCl2, CaCl2400 / NaBr, NaBr400 / MgCl2, NaBr50 / NaCl, NaBr400 / NaCl, CaCl2400 / MgCl2, and NaCl400 / KCl. Handleable membranes were obtained with combinations of NaBr400 / CaCl2, CaCl2400 / NaBr, NaBr400 / MgCl2, NaBr400 / NaCl, and CaCl2400 / MgCl2, all tested at the S2 molar ratio. Among the salts tested, the salts NaBr, CaCl2, and MgCl2 enabled the formation of handleable and stable albumin materials. Indeed, albumin membranes were obtained when the salt combination contained at least one salt that enabled membrane formation and the salt / albumin molar ratio of this salt was sufficient (>100). To assess the influence of the secondary salt on the material properties, the formed materials were characterized using relative yield, water absorption, and initial swelling. Figure 20 shows a membrane formulated with the CaCl2400 / NaBr combination. NaBr molar ratios ranging from 100 to 1000 were tested, and a control was prepared with only CaCl2 and BSA at a salt / albumin molar ratio of 400. The addition of NaBr to the formulation did not change the relative yield of the formulation; however, it produced a significant increase in water absorption. Changes in film properties were also observed with other combinations. Additionally, the feasibility of formulations with three salt combinations was also tested.These experiments showed that stable and easy-to-handle membranes can be produced with combinations of NaBr / CaCl2 / MgCl2 (molar ratios tested: 100 / 100 / 100, 200 / 200 / 200, 300 / 300 / 300). In conclusion, the use of multiple salts does not prevent membrane formation and provides a suitable tool for fine-tuning the material properties of Albupad.

[0124] Protein Investigation. Four batches of human serum albumin (HSA) were tested: HSAFAF (fatty acid-free), HSALFP (low-folate powder), HSARGP (reagent-grade powder), and rHSA (recombinant HSA from rice). Additionally, two globular proteins, γ-globulin (human origin) and hemoglobin (human origin), were tested. Protein solubility is a key criterion for the applicability of Albupad technology, and these globular proteins were selected for their suitable water solubility. Each protein was tested with NaBr, NaCl, and CaCl2 salts (salt / protein molar ratios: 0–2000). The protein and salt solutions were evaporated in an oven at 37°C for 7 days. The dried material was then washed and soaked in water for 48 hours to remove the salts, and water-insoluble materials were selected. Water-insoluble films were obtained for all human albumin proteins, including those with NaBr and CaCl2. These films were stable and easy to handle. γ-globulin-based films formed well with NaBr. Thus, Albupad technology can be used to produce stable, handleable materials using human serum albumin, recombinant human albumin, and human gamma-globulin.

[0125] 2.2 Loading of active substance Due to albumin's innate ability to load various substances, albumin-based materials are promising for drug delivery and sustained release. Furthermore, Albupad technology allows for loading of active substances before the formation of the material in an initial solution (preloading) and after formation (postloading). Preliminary studies have demonstrated that Albupad We demonstrated that the material could be preloaded with lipophilic substances, such as piroxicam and fluticasone, as well as the water-soluble molecule chlorhexidine. This investigation resulted in the formation of stable, handleable materials, validating the applicability of this loading strategy to Albupad technology. Furthermore, to establish proof of concept and study the time-dependent release of these substances, we also loaded the Albupad material with doxorubicin (DOX), an antitumor drug, and insulin (INS), a peptide used to treat diabetes. The loading of these substances was assessed by fluorescence imaging. Quantification of drug release in water was performed by time-dependent fluorescence titration.

[0126] Loading and release of preloaded DOX into Albupad membranes. BSA / NaBr and BSA / CaCl membranes were preloaded with DOX by incorporating DOX directly into the solution before evaporation, as shown in Figure 21. Different amounts of DOX were incorporated into the solution: 0.25, 0.5, 0.75, and 1 mg of DOX per membrane (membrane mass ≈ 400 mg). After evaporating the solution, handleable membranes were obtained. The prepared membranes appeared colored due to the presence of DOX. Furthermore, the intensity of the membrane coloration was proportional to the amount of DOX loaded. CLSM imaging was performed to visualize the presence of DOX within the membranes (Figure 22). In the BSA / NaBr membrane, DOX fluorescence was uniformly distributed within the membrane matrix (thickness = 500 μm), indicating successful loading of the active substance into the material (Figure 22). For the BSA / CaCl membrane, the opacity of the material prevented observation of the DOX loaded within the material matrix. However, DOX fluorescence was observed on their surfaces, revealing that DOX loading into these membranes was also successful (Figure 22).

[0127] The membranes were then washed with water to remove salts. The BSA / NaBr membrane was washed with water (3 × 20 mL) for 2 h. The BSA / CaCl2 membrane was washed with water (4 × 20 mL) for 2 h. The rinse solutions were collected, and the amount of DOX removed was measured by fluorescence spectroscopy at 485 nm (Figure 23). The DOX removed ranged from 20% to 30% for the BSA / NaBr membrane and from 35% to 50% for the BSA / CaCl2 membrane. Therefore, 50% to 70% of the DOX initially loaded remained in the membrane after washing.

[0128] DOX release from the membranes was studied in water at 37°C with stirring for 35 days. DOX titration of the supernatant over 35 days revealed a slow, controlled release profile of this active substance from the Albupad material with limited burst effect (Figure 24). Furthermore, 10-50% of the DOX present remained unreleased after 35 days, depending on the formulation, demonstrating the potential of the material for drug delivery.

[0129] Loading and release of INS-FITC preloaded onto Albupad membranes. The BSA / NaBr and BSA / CaCl2 membranes were preloaded with INS-FITC by incorporating INS-FITC directly into the solution before evaporation. The amount of INS-FITC incorporated into the solution was 0.25 mg per membrane (membrane mass approximately 400 mg). After evaporating the solution, handleable membranes were obtained. CLSM imaging was performed to visualize the presence of INS-FITC within the membranes. In the BSA / NaBr membrane, INS-FITC fluorescence was uniformly distributed within the membrane matrix (thickness = 500 μm), indicating successful loading of the active ingredient into the material (Figure 25). For the BSA / CaCl2 membrane, the opacity of the material prevented observation of the INS-FITC loaded within the material matrix. However, fluorescence was observed on their surface, revealing successful loading of DOX into these membranes (Figure 25).

[0130] The membranes were then washed with water to remove salts. The BSA / NaBr membrane was washed with water (3 x 20 mL) for 2 hours. The BSA / CaCl2 membrane was washed with water (4 x 20 mL) for 2 hours. The washed solution was collected, and the amount of removed INS-FITC was measured by fluorescence spectroscopy at 495 nm (Figures 2.7A and 2.7B). For the BSA / NaBr and BSA / CaCl membranes, 8% and 1% of the loaded INS-FITC, respectively, were removed during the washing process. Thus, after the washing process, more than 90% of the INS-FITC was present in the membranes. Subsequently, the release of INS-FITC from the membranes was investigated in water at 37 °C with stirring for 30 days. In accordance with the results obtained with DOX, titration of INS-FITC in the supernatant over 30 days revealed that the release profile of this active substance from the Albupad material was slow and controlled, with limited burst effects (Figure 2.7C). Furthermore, 60–80% of the available INS-FITC remained unreleased after 30 days, depending on the formulation, demonstrating the potential for sustained release of this active substance.

[0131] In conclusion, considering all the experiments performed, the feasibility of loading materials using the preloading method was proven. Furthermore, other active substances, such as gentamicin, were also successfully loaded into Albupad membranes at loading rates as high as 30% (wt / wt). Furthermore, the maximum loading rate depends on the solubility of the active substance and its interaction with albumin. Therefore, further investigations should enable the study and optimization of the loading of targeted active substances. Furthermore, the presence of proteases in the release medium should allow for increased substance release from the membrane.

[0132] 2.3 Preliminary evaluation of biocompatibility and in vivo biodegradability

[0133] Optimization of sterilization of Albupad material using electron beam irradiation. Prior to in vivo implantation of Albupad material, sterilization using electron beam irradiation was tested to verify its compatibility with the material and thus to prevent degradation or significant changes in its properties or the structure of the albumin that constitutes it. Two types of membranes were prepared: BSA / NaBr and BSA / CaCl2. These samples were irradiated in groups to test their stability under irradiation under the following conditions: with or without a radioprotectant (vitamin C), and in dry or hydrated (in water) membrane form. Four irradiation doses were tested: 5, 10, 25, and 25 kGy. The physical aspects, mass loss, and water uptake of the irradiated membranes were compared with those of a non-irradiated control batch. Furthermore, the IR spectra of the irradiated membranes were compared with those of the control to detect changes in the secondary structure of albumin. These experiments revealed that irradiation of the materials did not cause their degradation at all tested irradiation doses and that the use of vitamin C as a radioprotectant was not necessary. Comparison of irradiated samples with control batches showed no significant changes in the material's properties after irradiation. Furthermore, FT-IR analysis showed that irradiation of the material did not alter the amide I band and therefore did not damage the secondary structure of albumin in both dry and hydrated samples. In conclusion, sterilization by electron beam irradiation is compatible with Albupad materials and can be used efficiently at 25 kGy to sterilize these materials prior to in vivo evaluation.

[0134] Implant preparation, sterilization, and subcutaneous implantation in mice. To conduct a preliminary investigation of the biodegradability and biocompatibility of the AlbuPad material in vivo, the following batches of cylindrical implants were prepared: BSA / NaBr (initial BSA concentration = 300 mg / mL, initial NaBr concentration = 1.8 M, n = 5), BSA / CaCl2 (initial BSA concentration = 200 mg / mL, initial CaCl2 concentration = 2.1 M, n = 10), HSA / NaBr (initial HSA concentration = 300 mg / mL, initial NaBr concentration = 1.8 M, n = 5), and HSA / CaCl2 (initial HSA concentration = 200 mg / mL, initial CaCl2 concentration = 2.1 M, n = 10). A control batch of implants was prepared by crosslinking HSA in the presence of glutaraldehyde (HSA / Glu, n = 5). The grafts were then thoroughly washed and cut to the recommended size (length ≈ 1 cm, diameter ≤ 5 mm) (Figure 27A). The water absorption rates of these materials were used to compare batches prepared with BSA and HSA. BSA / NaBr and HSA A / NaBr had similar visual profile and water absorption values ​​(124±3% and 127±21%, respectively), as did BSA / CaCl2 and HSA / CaCl2 (224±52% and 220±10%, respectively) (Figure 27B). The grafts were then dried in 24-well plates at 37°C for 24 hours. The dried batch of grafts was then sterilized using the method of choice (electron beam, 25 kGy).

[0135] The sterilized grafts were then used for subcutaneous implantation in mice. The grafts were rehydrated in PBS at 4°C for 6 hours and rinsed with PBS before implantation. Swiss mice were implanted with BSA / CaCl2, HSA / CaCl2, and HSA / GLU grafts (three mice per graft type). Nude mice were implanted with BSA / NaBr, BSA / CaCl2, HSA / NaBr, and HSA / CaCl2 grafts. Acute toxicity was observed in the control group implanted with the BSA / GLU material, and mice in this group were sacrificed. Mice implanted with the Albupad material survived throughout the experiment (28 days) without significant weight fluctuation (±5-10%). Graft volume was measured through the skin using a vernier caliper. In the Swiss mouse group, BSA / CaCl2 implants were completely degraded after 17 days of implantation, whereas HSA / CaCl2 implants were not completely degraded by the end of the experiment at 28 days, losing an average of 50% of their volume. In nude mice, degradation of BSA / NaBr, HSA / NaBr, BSA / CaCl2, and HSA / CaCl2 was 23%, 35%, 53%, and 43% of their volume, respectively, after 28 days of implantation. After sacrificing the mice on day 28, histological evaluation of sections of the implants and surrounding tissues was performed by microscopic examination after Gomori and Picrosinius staining. Observation of the prepared cuts revealed that the implants were not fragmented, indicating that their biodegradation was progressive and did not result in the formation of large fragments (Figure 28A). The phenotype of the tissue surrounding the implants was normal, and the formation and orientation of collagen fibers were observed to be similar to those observed in native tissue (Figure 28B). No fibroplasia was observed in contact with the materials. Thus, the implants tested were proven to be non-toxic to the surrounding tissue or to the mice and biodegradable in vivo at rates dependent on the type of implant. Experimental Section

[0136] Chemical Reagents. Bovine serum albumin (fraction V, ≥96%) was purchased from Acros Organics. Hemoglobin (human), gamma-globulin from bovine blood (≥99%), recombinant human albumin (rHSA, expressed in rice), sodium bromide (NaBr), potassium chloride (KCl), doxorubicin (DOX), insulin conjugated to FITC (INS-FITC), and deuterium oxide (DO) were purchased from Sigma-Aldrich. Sodium chloride (NaCl) was purchased from VWR Chemicals. Magnesium chloride (MgCl, anhydrous) was purchased from Fluka. Calcium chloride (CaCl, 2H2O) was purchased from Merck. The following human serum albumin (HSA) was purchased from SeraCare: HSA reagent grade powder (RGP), HSA low folate powder (LFP), and HSA fatty acid free (FAF).

[0137] Formulation (General Procedure): A solution of BSA (100 mg / mL) and NaBr 1 M was prepared in sodium acetate buffer (0.2 M) at pH 6. This solution was poured into a silicone mold and allowed to evaporate at 37 °C until dry. The resulting dried biomaterial was washed to remove salts and immersed in distilled water at room temperature for 48 hours. The water-insoluble film (BSA / NaBr) was then collected and characterized.

[0138] Initial characterization. The formulated membranes were compared using relative yield (Eq. S1), water uptake (Eq. S2), and initial swelling (Eq. S3). BSA represents the initial mass of albumin used in the formulation. i represents the area of ​​the circular vessel used during the evaporation process. W d represents the mass of the final dry membrane after washing with distilled water for 48 h and drying overnight in a 37 °C oven .V d is the volume of the dry film measured by immersing the material in distilled water at room temperature. h A represents the mass of the hydrated film at equilibrium after immersion in distilled water for 24 hours and removal of excess surface water using filter paper. his the area of ​​the surface of the hydrated film in equilibrium after immersion in distilled water for 24 h, calculated after measuring the diameter using an electronic digital caliper (TACKLIFE-DC01, accuracy ±0.2 mm).

[0139]

number

[0140] Scanning electron microscopy (SEM). Scanning electron microscopy and microanalytical evaluations were performed using a Quanta 250 FEG SEM (FEI Company, Eindhoven, The Netherlands) operating at an accelerating electron voltage of 10 kV. For SEM experiments, the BSA / NaBr664 films were dried and then coated with a gold-palladium alloy using a Hummer Jr sputtering device (Technics, Union City, CA, USA).

[0141] Stability in organic solvents. BSA / NaBr membranes were placed in 5 mL of the following organic solvents: DMSO, acetonitrile, and dichloromethane. The membranes were then incubated at room temperature with stirring for 72 hours. The membranes were then washed with water and their mass loss was characterized (Eq. S4).

[0142]

number

[0143] Mechanical properties of the membranes. Compression tests were performed using a Kinexus ultra+ rheometer (Malvern, United Kingdom) in a parallel plate configuration (20 mm diameter). Three hydrated batches of BSA / NaBr membranes (in distilled water) (thickness = 0.7 mm) were used. The elastic modulus (E) was calculated within the elastic region of the strain (ε)-stress (σ) curve (Equation S5). σ=E×ε (S5)

[0144] Rheological measurements. The viscoelastic behavior of BSA / NaBr films (thickness = 0.7 mm) was evaluated using a rheometer Kinexusultra+ (Malvern, United Kingdom) in a parallel plate configuration (diameter 20 mm). The films were pre-hydrated with distilled water for 24 h. A BSA / NaBr disk was loaded between the plates, and the gap was closed until the sample was in good contact with both plates (normal force <1 N). At the beginning of the experiment and between experiments, the samples were allowed to equilibrate for 5 min. During this time, the normal force decreased to values ​​below 0.1 N for all samples. Amplitude sweep tests were performed at 25 °C with a fixed frequency of 0.5 Hz, ranging from 0.01 to 100% shear strain. The shear modulus G * (ω)=σ(ω) / γ(ω) is obtained from the ratio of stress (σ) to strain amplitude (γ). G * (ω) = G´ + iG” is the complex number of the elastic storage modulus (G´) and the viscous loss modulus (G”). The absolute value of the shear modulus |G * |, |G * |=(G´ 2 +G” 2 ) 0.5 was calculated using

[0145] Contact angle measurements were performed using an Attension Theta tensiometer and Biolin Scientific OneAttension software (Sweden). Experiments were performed in "sessile drop" mode to measure the static contact angle of water on dried BSA / NaBr films. A Hamilton microsyringe (needle diameter = 0.7 mm) was used to dispense water droplets. Each measurement was performed using a 5 μL drop of milliQ water (droplet velocity = 2 μl / s) and was repeated three times for each sample. The built-in camera captured a series of images of the sessile drop (frame rate = 14 fps) while measuring the tangent angle made by the drop at its intersection with the material. The average contact angle (average of the left and right contact angles) was recorded after 10 s, sufficient time for the drop to stabilize.

[0146] IR analysis. FTIR experiments were performed on a Vertex70 spectrometer (Bruker, Germany) using a DTGS detector. Spectra were analyzed at 2 cm using Blackman-Harris three-term apodization and Bruker OPUS / IR software (version 7.5). -1 128 interference images with resolution from 800 to 4000 cm -1 The spectra were recorded in Attenuated Total Reflection (ATR) mode using a single-reflection diamond ATR by averaging over 1000 nm. The prepared BSA / NaCl film (dried) was finely ground and the spectra were recorded in the amide I band region (1700–1600 cm). -1 Data processing was performed using OPUS 7.5 software (Bruker Optik GmbH) to decompose the amide I bands. Prior to curve fitting, the spectra were baseline-corrected in the amide I band region and normalized using the "min-max" method of normalization. The number of subbands and their positions were determined from the fourth derivative of the spectrum. Deconvolution was performed according to a least-squares iterative curve-fitting program (Levenberg-Marquardt) using Gaussian line shapes. In the final fit, the height, width, and position of all bands were adjusted to reduce the residual RMS error as much as possible (below 0.005), without changing at least one of these parameters each time. Finally, the accuracy of the curve fit was confirmed by comparing the second derivatives of the original and fitted curves. The fractional areas of the fitted components were used to calculate the percentages of various secondary structural elements (α-helix, β-sheet, β-turn, and random coil) after identifying them according to the literature.

[0147] Preloading formulation. BSA solution (100 mg / mL) was prepared in pH 6 acetate buffer (0.2 M) and mixed with NaBr (NaBr / BSA molar ratio = 400) or CaCl2 (CaCl2 / BSA molar ratio = 700) salt. For doxorubicin (DOX) preloading, the following amounts of DOX were added to the initial solution before evaporation: 0.25, 0.5, 0.75, and 1 mg per membrane (membrane mass = 400 mg). For insulin (INS-FITC) preloading, 0.25 mg of INS-FITC per membrane (membrane mass = 400 mg) was added to the initial solution before evaporation. The solution was placed in a non-stick silicone mold and allowed to evaporate for 48 hours at 37 °C. The resulting dried biomaterial was thoroughly washed with 3 × 20 mL of water for BSA / NaBr membranes and 4 × 20 mL of water for BSA / CaCl2 membranes to remove salts.

[0148] Confocal laser scanning microscopy characterization (CLSM). To visualize the presence of DOX or INS-FITC in the preloaded membranes, CLSM imaging was performed using a ZEISS LSM710 confocal microscope. The excitation / emission wavelengths for membrane imaging were fixed at 450 / 515 nm for both preloaded DOX and INS-FITC materials.

[0149] Release experiments. The release of DOX or INS-FITC preloaded membranes was monitored with a Genius XC spectrofluorometer (SAFAS, Monaco). Release experiments were carried out in HO (10 mL per membrane) at 37 °C. After each supernatant was collected, 10 mL of fresh water was added. The supernatants were analyzed with the spectrofluorometer. The excitation and emission wavelengths of DOX and INS-FITC were λ , respectively. ex / λ em = 485nm / 595nm and λ ex / λ em =495nm / 520nm.

[0150] Statistical analysis. Data were analyzed using R (version 3.6.1, R Foundation for Data were analyzed using the Statistical Computing Center (Statistical Computing, Vienna, Austria). Normality of distribution was determined with the Shapiro-Wilk test. Equality of variances was determined with an F-test. If data were normally distributed and sample variances were equal, two means were compared using a two-tailed t-test. If these two conditions did not apply, a Mann-Whitney test was performed instead. Values ​​were considered statistically significant at p<0.05.

Claims

1. A process for preparing an insoluble, manageable biomaterial, said process comprising: a) at least one serum protein selected from albumin and globulin, and NaBr, NaI, KI, CaCl ２ , MgCl ２ , K.C. ２ H ３ O ２ and N.H. ４ HCO ２ wherein the serum protein and the salt have a molar ratio (salt / protein) of 200 to 2000, and the M / S ratio, which is the ratio between the weight of the serum protein and the surface area of ​​the container, is 10 mg / cm ２ ~400 mg / cm ２ preparing a solution comprising: b) evaporating the solution obtained in step a) neat, as a foam obtained by foaming the solution obtained in step a), or as a mixture thereof, at a temperature comprised between 4 and 50°C at atmospheric pressure, or at a lower temperature under vacuum, or at a pressure lower than atmospheric pressure, until a solid phase of two immiscible phases is formed, or until a substantially dry solid is obtained; c) washing the solid phase or the dried solid obtained in step b), A process by which an insoluble, manageable biomaterial is obtained.

2. 2. The process of claim 1, wherein the dried solid obtained in step b) is washed until at least 90% by weight of the at least one salt is removed.

3. 2. The process of claim 1, wherein the albumin is selected from human serum albumin, bovine serum albumin, porcine serum albumin, ovalbumin, vegetable albumin, or recombinant albumin.

4. The process according to any one of claims 1 to 3, wherein at least one additive is added during step a) and / or step b).

5. 5. The process according to claim 4, wherein the at least one additive is selected from among polymers, in particular uncharged, positively charged, negatively charged and zwitterionic polymers, non-ionic amino acids and particles.

6. A biomaterial obtainable by the process defined in any one of claims 1 to 5.

7. 7. The biomaterial of claim 6, wherein the percentage of secondary structure of the protein is at least the same as that of the corresponding native protein, as determined by IR analysis or small-angle X-ray scattering.

8. 8. Biomaterial according to claim 6 or 7, characterized in that at least one active ingredient is incorporated into the biomaterial.

9. 9. The biomaterial according to claim 6, wherein the at least one active ingredient is selected from the group consisting of anti-cancer substances, anti-inflammatory agents, immunosuppressants, immunomodulators, cell growth inhibitors, anticoagulants, antithrombotic agents, modulators and inhibitors of cell-extracellular matrix interactions including enzymes, analgesics, antiproliferative agents, antifungal substances, cytostatic substances, growth factors, enzymes, hormones, steroids, non-steroidal substances, and antihistamines.

10. Use of the biomaterial according to any one of claims 6 to 9 as a support for in vitro tissue engineering and / or for in vitro cell culture and in vitro proliferation.

11. A biomaterial as defined in any one of claims 6 to 9 for use as a drug.

12. 12. The biomaterial of claim 11 for replacing defective tissue in a subject in need thereof or for use as a drug-releasing formulation.

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